Chapter 21 Diseases associated with viruses and Chlamydia – I
VIRAL DISEASES WITH MANIFESTATIONS ATTRIBUTABLE TO INVOLVEMENT OF THE BODY AS A WHOLE 1157
ENZOOTIC BOVINE LEUKOSIS (BOVINE LYMPHO SARCOMA) 1209
BOVINE IMMUNODEFICIENCY-LIKE VIRUS 1221
VIRAL DISEASES CHARACTERIZED BY ALIMENTARY TRACT SIGNS 1223
The comments made earlier about diseases associated with bacteria and their importance as infectious diseases to animal agriculture apply also to diseases associated with viral infections, which are presented in this chapter and Chapter 22. However, there are several factors that make viral diseases even more important.
Viruses have a much greater capability for surviving independently of their host. Spore-forming bacteria survive for long periods in the environment, but, in general terms, viruses are more viable at large than are bacteria. Because of their structure, viruses are much less susceptible to destruction by cleaning and disinfectant agents than are bacteria.
Viruses also have a greater capacity for living in relative harmony with the host, without destroying it. Some, such as the Lentivirus group, produce persistent infections with antigenic drift and the emergence of novel antigenic strains, with consequent relapsing disease. Many of the viruses are insect-borne and have reservoir hosts in other species, especially wildlife. These properties pose unique problems for the diagnosis, control and eradication of these viral infections, and in the development of suitable vaccines. In terms of exotic disease threats, viral diseases can represent a much more significant challenge than bacterial diseases. They have the capacity for escaping beyond standard prophylactic barriers and proceeding on a path of global disease. In recent times African swine fever, bluetongue and African horse sickness have made territorial gains in this way, while the vesicular diseases and the insect-borne encephalitides are constantly on the move into new territory. Perhaps the biggest threat, because of its zoonotic potential, is Rift Valley fever. The viral diseases with potential for severe economic effect and risk for spread to uninfected areas are listed in the table of List A diseases (Table 16.1).
The role of the field veterinarian is made very much more onerous by the presence of these diseases. Because of the speed with which they spread, for example when foot-and-mouth disease virus is airborne, or when ephemeral fever is carried by Culicoides sp., they can appear for the first time in an outbreak some distance from their point of entry and in places where an imported exotic disease would not be expected to appear for the first time. Field veterinarians must be on the alert at all times for the appearance of new diseases in their practice areas, and be aware of the implications of their presence, and the kinds of emergency action necessary when they are recognized.
There is no way to prepare oneself for an encounter with an exotic viral disease other than by becoming familiar with their clinical and clinico-pathological findings and their epidemiological behavior. This chapter and Chapter 22 attempt to fill that need.
Hog cholera, also known as classical swine fever (CSF), is a highly infectious pestivirus infection of pigs which causes acute, chronic or inapparent disease. At one time it was characterized clinically by an acute highly fatal disease and pathologically by lesions of a severe viremia. It is now known that chronic or inapparent disease also occurs, including persistent congenital infection in newborn pigs infected during fetal life. In many countries, where it is endemic, clinical ability will diagnose it more often than laboratory skills, as long as you remember that is a real diagnostic possibility.
Hog cholera virus, a pestivirus belonging to the genus Flaviviridae and related to the bovine virus diarrhea virus
Affects domestic pig of all ages; causes major economic losses interfering with trade when outbreaks occur in pig-raising countries. Occurs in Europe, South America and the Far East. Highly virulent virus causes high morbidity high mortality; less virulent strains cause milder form. Transmitted by direct contact, feeding of uncooked pork products. Neutralizing antibodies provide protection
Sudden onset of peracute deaths first indication in herd. Many pigs affected within days. Severe depression, fever, anorexia, purplish discoloration of skin, ocular discharge, nervous signs, and death in few days. Nervous form may predominate. Reproductive failure in pregnant sows (abortions, mummification, stillbirths, birth of persistently infected pigs)
Hog cholera is associated with a virus of the family Flaviviridae, genus Pestiviridae. There is only one antigenic type but a number of strains of variable virulence and antigenicity.1,2 Genetic typing has been described in detail3,4 and is absolutely essential for following the world wide pattern of infection.5 For example, there are at least 8 antigenic groups in Thailand. It is generally stable in vitro and in vivo.6 The viruses have been grouped. In Europe they used to be mainly group 1 viruses but these have been replaced by group 2 viruses. Group 1 still survives in Russia and also Cuba. Group 3 appear to be found in Asia only.
The virus has an antigenic relationship to the bovine virus diarrhea virus (BVDV).7 In Denmark, BVDV antibodies were found in 6.4% of the sera of pigs while all area were found to be free from antibodies to the hog cholera virus.
The pig is the only domestic animal species naturally infected by the virus. All breeds and ages are susceptible and adults are more likely to survive an acute infection. The disease originated in the United States but is now almost worldwide in distribution. Canada, Australia, New Zealand and South Africa have not experienced the disease for many years. A mild form of the disease occurred in Australia in 1960–1961.8 The disease was eradicated from the United Kingdom during the period 1963–1967 and the United States was declared free of the disease in 1978. Outbreaks of acute hog cholera have occurred occasionally in other countries but were quickly controlled by a rigorous policy of slaughter and quarantine. The disease occurred in the United Kingdom in 1986 in which three primary outbreaks were identified; all outbreaks were attributed to the feeding of unprocessed waste feed containing imported pig meat products. A similar origin was suspected for the outbreak in the UK in 2000.9 In this outbreak an interesting feature was the transport of infected pig carcasses from a site where bodies were dumped for quite long distances by scavenging foxes, infecting new outside pig arks across fields as they went. Between 1982 and 1984 epidemics occurred in Germany, the Netherlands,10 Belgium, France, Italy, Greece, and the Iberian peninsula. As of 1985, six countries in Europe were free of classic swine fever: Denmark, Ireland (including Northern Ireland), Norway, Sweden, Finland, and Switzerland.
The disease is currently endemic in most countries of South America and the Far East except Japan and Korea. In Asia the problem is the back-yard pig that is not vaccinated and is always a reservoir. Here extension services, appropriate vaccination schemes and regulatory control is difficult to implement but it may be time to try for world wide eradication. The costs are astronomical (the Belgian outbreak of 1997 cost an estimated 11 million euros,11 and the Netherlands outbreak even more).12 This 1997–1998 outbreak in the Netherlands was very serious.13 The disease has also concentrated in certain parts of Europe where the pig populations are intense and live in close proximity to wild boar and feral pig populations. For instance outbreaks occurred regularly between 1997–2001 in Croatia, one source was imported pig meat and the other strains reaching domestic pigs from wild boar.14 These areas include parts of Germany and Poland and probably most of Eastern Europe. The disease together with ASF is endemic in the central highlands of Sardinia. Many outbreaks of classical swine fever occurred in Germany between 1993 and 1995 and major outbreaks occurred in 1996–1997 in Germany and the Netherlands.15 The risk factors for Germany have been described.16 In these countries, over the past 25 years, the disease occurred as a series of epidemics in which many swine herds in a geographical area were affected within a few months. As pig production continues to become intensified the disease has been more difficult to control. In some areas of Western Europe, the pig population has more than doubled in the last 10 years. The hazards of introduction of the disease have increased considerably, due to the introduction of free movement of animals and the non-vaccination policy currently practiced in the European Union. Improvements in the Identification and Recording system which is supposed to identify all relevant animals in a particular population and record their movements and changes in inventory, are necessary to support the control of contagious diseases such as classical swine fever.17 Most of the outbreaks of swine fever in Belgium in 1990 had to be classified (afterwards) as being due to ‘area’ and ‘unknown’ transmission.
In the Philippines, the disease is endemic on many large-scale swine farms.18 In spite of vaccination of the sows and boars every 6 months and piglets at 6–8 weeks of age, the disease causes sub-optimal performance in 10–30% of pigs between 7–16 weeks of age.
Infection with the classical swine fever virus has also occurred in the wild boar population in Tuscany in Italy, Germany, France, Austria, and Czechoslovakia19 and Croatia. Serological surveys of wild boar in Sardinia found an overall prevalence of 11%20 and seropositive boars were found not only in areas where they share their habitat with free ranging domestic pigs but also in areas of the island where contacts between wild and domestic pigs are unlikely to occur. Thus there may be transmission and persistence of the virus within the wild boar population. This has occurred with the low virulence strain in Germany.21 The persistence of infection in a wild boar population in the Brandenburg region of Germany provided optimum conditions for the establishment of a CSF epidemic in Germany.22
The disease usually occurs in epidemics, often with a morbidity of 100% and a case–fatality rate approaching 100%, when a virulent strain of the virus infects a susceptible population. However, in recent years outbreaks of a relatively slowly spreading, mild form of the disease have caused great concern in many countries. The disease associated with strains of low virulence may be unnoticed in growing and adult pigs but the infection can be associated with perinatal mortality, abortions and mummified fetuses. In a recent outbreak a mild form given experimentally to sows only produced a mild viremia with widespread antigen distribution, but without clinical signs, except lesions of hemorrhagic dermatitis. It did however produce an antibody response and transplacental infection.23
The source of virus is always an infected pig or its products and the infection is usually acquired by ingestion but inhalation is also a possible portal of entry. Direct animal to animal contact is the most important method of spread. Infected pigs shed a large amount of the virus in all normal secretions24 – nasal, salivary, urinary, fecal and is important in transmission when there are clinical signs of the disease. It is excreted in the urine for some days before clinical illness appears and for 2–3 weeks after clinical recovery. Virus spread via excretions is more important in early stages of an outbreak.25 Highly contagious by direct contact, it is likely to be transmitted by aerosol only when all the pigs in the same airspace are viremic26 and even then only for a distance of 1 meter.27 It has been spread experimentally by aerosol which followed the pattern of air currents.28
Sick pigs excrete virus until they die, and obviously even longer if they recover. The resistance and high infectivity of the virus make spread of the disease by inert materials, especially uncooked meat, a major problem. The UK virus in the 2000 outbreak probably came from an infected pork product, imported illegally and fed to an outdoor pig.29 Outside pens, in warm weather and exposed to sunlight, lose their infectivity within 1–2 days. The ability of the virus to survive in the environment in more favorable situations is uncertain. However, it is probable that it can survive for considerable periods as the virus is quite resistant to chemical and physical influences. Transmission from neighboring units is very easy.5 One of the major features of the recent Dutch outbreak was the proof that transmission from boar studs (AI) was possible as infected boars excreted virus in semen30-32 and the virus probably infects spermatogonia.33 It was shown that following insemination with semen containing CSF antibodies could occur as early as 7 days and all pigs were positive by 14 days.34 The transmission rates in the Dutch outbreak have been calculated.35
In areas free of the disease, introduction is usually by the importation of infected pigs or the feeding of garbage containing uncooked pork scraps. Hopefully, in Europe the ban on swill feeding will prevent further cases of infected meat causing the problems.5 Movement of pigs which are incubating the disease or are persistently infected is the most common method of spread. The infection usually originates directly from infected breeding farms. Birds and humans may also act as physical carriers of the virus. In endemic areas, transmission to new farms can occur in feeder pigs purchased for finishing, or indirectly by flies and mosquitoes, or on bedding, feed, boots, automobile tires or transport vehicles. Farmers, veterinarians and vaccination teams can transmit the virus by contaminated instruments and drugs10 but recent evidence suggests that mechanical transmission may have been overestimated.25 Farmers can spread the virus within a herd by treating sick animals or employing routine health management procedures such as iron injections of newborn pigs. The common practice of not changing syringes and needles between farm visits constitutes a major risk when viremic animals are present. The most common cause of dissemination occurs through the movement and sale of infected or carrier pigs through communal sale yards when there is ample opportunity for infection of primary and secondary contacts.
When the disease is introduced into a susceptible population, an epidemic usually develops rapidly because of the resistance of the virus and the short incubation period. In recent years outbreaks have been observed in which the rate of spread is much reduced and this has delayed field diagnosis. It is not spread by dogs, cats or rats36 and bird transmission is unlikely.37
Following the 1997–1998 severe outbreak in the Netherlands analysis showed that there were five major increased risk factors identified.38 These were: (i) presence of commercial poultry on the farm; (ii) visitors to the units not being provided with protective clothing; (iii) drivers of lorries using their own clothes not the premises they were visiting; (iv) larger size; and (v) aerosols produced by high pressure hosing. Reduced risk was associated with (i) over 30 years experience of farming; and (ii) additional lorry cleaning before being allowed on to the farm.
Historically, infection with the hog cholera virus rapidly resulted in severe clinical disease. It is now recognized that with less virulent strains, a carrier state can occur, at least for a period of time. Following exposure to these strains, pigs may become infected without showing overt signs of the disease and although they may eventually develop clinical disease, this latent period is of importance in dissemination of infection when such pigs are sold and come in contact with others. In recent outbreaks in high pig density areas in Belgium, the interval between the first occurrence of clinical signs and the report of a suspect herd was shorter when the disease was first diagnosed in finishing pigs rather than in sows, boars or nursing piglets.39 The proportion of clinically affected animals was positively correlated with the proportion of serologically positive animals.
Susceptible pregnant sows, if exposed to less virulent strains of the virus, may remain clinically healthy but infection of the fetuses in utero is common and virus may be introduced into susceptible herds by way of these infected offspring. The sow with ‘carrier sow syndrome’ can give birth to normal healthy appearing piglets which are persistently infected and immunotolerant; these pigs along with those with chronic infections are responsible for the perpetuation of the virus in the pig population.10 A fully virulent virus may also be transmitted in this manner if the sows are treated with inadequate amounts of antiserum at the time of exposure or if they are exposed following inadequate vaccination. Piglets infected in utero, if they survive, may support a viremia for long periods after birth.
During outbreaks of classical swine fever in Germany between 1993 and 1995, differing clinical courses were observed ranging from mild clinical signs to severe typical disease. The genotype of pigs may influence the outcome of hog cholera virus infection. In certain pig breeds the chronic form of the disease is more likely to occur and these pigs may excrete the virus over prolonged periods.40 Experimental inoculation of purebred pigs resulted in acute fatal infections, while cross-bred pigs experienced acute, chronic and transient infections.40
The most virulent strains produce clinical disease in pigs of all ages. But there are differences in the clinical and pathological features between strains of the virus8 and in their virological characteristics.41 The less virulent strains cause only mild clinical disease or disease restricted primarily to fetal and newborn piglets. It is probable that this variance has always occurred in field strains of the virus but the use of inadequately attenuated live virus vaccines is also a contributory factor. The occurrence of variation in virulence and antigenicity has been recognized as a cause of failure of vaccination and ‘vaccine breakdowns’. It is equally important in causing problems with the diagnosis of hog cholera in eradication programs when infection is manifest in patterns not traditionally associated with this disease.
Genetic analysis of isolates of the virus for a series of epidemics of swine fever in Italy affecting both domestic pigs and wild boar has provided useful epidemiological information.42 The isolates were divided into three subgroups and it is suggested that there have been at least two separate introductions of classical swine fever over a 7-year period and that the virus has been transmitted between domestic pigs and wild boar. Molecular analysis can aid in tracing the transmission of the virus from domestic pigs to wild pigs and back to domestic pigs.
In the outbreaks of hog cholera in England in 1986, affected pigs in the first outbreak exhibited clinical signs and necropsy lesions indicative of a virulent strain of the virus. However, in subsequent outbreaks, clinical disease was much milder and case–fatality rates low. Experimental infection of pigs with a field isolate of the virus resulted in variations in clinical response, from acute illness to inapparent infection, including minimal changes visible at necropsy, all of which indicates that genotype may influence the pathogenesis of the disease. High titers of virus were found in several tissues of one experimental pig which was recovering, even in the presence of serum neutralizing antibodies. It is clear that some infected pigs may pass through an abattoir without detection because of the absence of lesions.
The virus is destroyed by boiling 5% cresol, or 3% sodium hydroxide and by sunlight, but it persists in meat which is preserved by salting, smoking and particularly by freezing. The virus can be inactivated in at least 80% of pork hams after exposure to a flash temperature of 71°C (159°F). It can survive in infected uncooked ham pork for at least 84 days and 140 days in diced ham or sausage43; bacon for 27 days after traditional curing processes and for at least 102 days in hams cured in salt concentrations of up to 17.4%, which is much higher than that normally used in curing bacon. The use of lower salt concentrations in curing solutions, and the decreased time between slaughter and consumption as a result of modern abattoir practices, increases the risk of disease transmission. It survives pH ranges from 3 to 11. Persistence in frozen meat has been observed after 4.5 years. The virus persists for 3–4 days in decomposing organs and for 15 days in decomposing blood and bone marrow.
Maternal antibodies may interfere with the production of viral specific cell mediated immunity.44 Neutralizing antibodies occur as early as 9 days after infection in recovering pigs and after 15 days in fatally infected pigs.45 Neutralizing antibodies are the most important antibodies in terms of protection. The maximum antibody response occurs 3–4 weeks after infection and levels may persist indefinitely but last at least 6 months. In chronic hog cholera, neutralizing antibodies may be transiently detectable during the phase of partial recovery between 3 and 6 weeks after infection. Low virulent strains of hog cholera may cause inapparent infections and are described as poorly immunogenic but in some instances may induce considerable titers of neutralizing antibodies in immunocompetent pigs. Cellular immunity mechanisms are probably very important in that it has been shown that there is CSFv specific IFN-gamma formed early after antigen exposure.46 These mechanisms produce a higher response after I/N or oral vaccination than after I/M vaccination and therefore vaccines should be looked at for their potential to induce higher T-cell responses.47 Intra-uterine infection of piglets with the virus may induce a state of specific immunological unresponsiveness. The piglets are persistently viremic and may continue to live for several weeks or months but the majority die within the first 3 weeks of life. Piglets with PRRSv infections have been shown to produce a poorer response.48
Hog cholera has been responsible for large economic losses in the swine industry worldwide. It is considered to be the most important disease of pigs in the European Union and a common program of eradication in the member states is in effect. The magnitude of the economic importance of the disease is directly proportional to the size of the pig population and the standards of the swine industry. In countries with intensified systems of pig production, such as the Netherlands, it is estimated that the direct costs of transport and destruction of infected herds, disinfection of premises, indemnities to farmers, vaccination, and identification and registration of pigs on behalf of the control of the disease amounted to a large percentage of the gross slaughter value. The additional, indirect, damage as a result of loss of production on infected farms, standstill of pig movements in affected areas or regions and restrictions on export is difficult to evaluate. Losses due to the death of pigs are aggravated by the high cost of vaccination programs in enzootic areas and by the problem that vaccination may not be completely effective in controlling epidemics. Recovered or partially recovered pigs are very susceptible to secondary infections, and exacerbation of existing chronic infections such as enzootic pneumonia are likely to occur during the convalescent period. Between 1990 and 1994, outbreaks occurred in Belgium which resulted in the destruction of about 2 million pigs and the total cost of control by 1995 was $120 million.49
The tonsil is the primary site of virus invasion following oral exposure. Primary multiplication of the virus occurs in the tonsils, beginning within several hours after infection. The virus is first found in plasma before the mononuclear cell populations.50 The primary cell in the peripheral blood to be infected is the mixed granulocyte.51 The virus then moves through lymphatic vessels and enters blood capillaries, resulting in an initial viremia at approximately 24 hours. At this time the virus is present in the spleen and other sites such as peripheral and visceral lymph nodes, bone marrow and Peyer’s patches. The virus exerts its pathogenetic effect on endothelial cells, lymphoreticular cells and macrophages, and epithelial cells.52 A B-lymphocyte deficiency associated with viral destruction of germinal centers in lymphoid tissues is the most significant pathoimmunological consequence of acute hog cholera infection.53 This lymphocyte apoptosis which is activation induced programmed cell death is one of the key features of CSF infections.54
Most of the lesions are produced by hydropic degeneration and proliferation of vascular endothelium, which results in the occlusion of blood vessels. This effect on the vascular system results in the characteristic lesions of congestion, hemorrhage and infarction from changes in arterioles, venules and capillaries. Thrombosis of small and medium-sized arteries is another feature. Vascular changes are most severe in the lymph nodes, spleen, kidneys and gastrointestinal tract. Lesions related to the effects on the endothelial cells also occur in the adrenals, central nervous system and eyes. Atrophy of the thymus, depletion of lymphocytes and germinal follicles in peripheral lymphoid tissues, renal glomerular changes and splenitis are characteristic. A leukopenia is common in the early stages, followed by a leukocytosis in some animals, and anemia and thrombocytopenia occur.52 The thrombocytopenia may be caused by massive platelet activation and subsequent phagocytosis of platelets secondarily to the release of platelet activating factors by activated macrophages.55 Disseminated intravascular coagulation is common with microthrombi in small vessels, particularly of the kidney, liver, spleen, lymph nodes, lung, intestine, and intestinal lymph nodes. The end stage of a lethal infection in the natural host is associated with a marked depletion preferentially of B-lymphocytes in the circulatory system as well as in the lymphoid tissues.14 Macrophage activation and subsequent release of pro-inflammatory cytokines, plays an important role in the development of the classical signs of CSF. This is particularly true for the pulmonary intravascular macrophages.56
It has been shown that there is a significant expression of TNFα in virus infected lymph nodes.57 It may be that commitment to apoptosis may depend on the IFN production.58 In these lymph nodes lymphocyte death occurred by apoptosis and some of the cells were positive on IHC for both TNFα and apoptosis. It may be that the release of the TNFα may induce the apoptosis in the uninfected bystander cells. Early immunosuppression is an important feature of the development of CSF59 with the depression of CD1+, CD4+ and CD8+ common thymocytes. It has recently been shown that CSF can replicate in the dendritic cells and control IFN type 1 responses without interfering with immune reactivity.60 It is still not clear, even though it is known that there is clear targeting of macrophages and monocytes, how these cells produce this immunosuppression and account for the death of the T-lymphocytes.61 It is known that the dendritic cells are the sentinels of the immune system62 and respond to easy viral contact.63 They then develop the effective immune responses by migrating into the lymphoid tissue to present the processed viral antigens to the T-lymphocytes.64 However, in both CSF and BVD infections there is no activation of the dendritic cells65 and this may be a feature of pestivirus infections and enable them to evade the immune response. At the same time there is no interference with the maturation of the dendritic cells. The virus induces pro-inflammatory cytokine production (IL-1, IL-6, and IL-8) by 3 hours and even further at 24 hours post-infection and also increases the coagulation factors, tissue factor and vascular endothelium cell growth factor.66 Endothelial cells that were chronically infected were unable to produce IFN type 1 and these cells were also protected from apoptosis. This establishes a long-term infection of endothelial cells with virus replication and increasing levels of IL-1, IL-6, and IL-8. It shows that there has been long term interference with cellular antiviral defences67 possibly by targeting interferon regulating factor 3 like BVDv does68,69 or by increased binding of NF-κβ (kappa/beta) which modulates an apoptotic pathway controlling several anti-apoptotic genes.70
In many cases, secondary bacterial infection occurs and plays an important part in the development of lesions and clinical signs.
The experimental disease is characterized by a biphasic temperature elevation at the 2nd and 6th day after inoculation, a profound leukopenia and an appreciable anemia 24 hours after inoculation, diarrhea at the 7th day, and anorexia and death on the 4th to 15th day in slaughter pigs.71 The anemia can be explained by the infection of 2–9% of the megakaryocytes 2–9 days after infection.72
The inoculation of pregnant sows with a low-virulent field strain of hog cholera virus at various stages of pregnancy results in prenatal mortality in litters from sows infected at pregnancy day 40 and postnatal death at 65 days. The later that infection occurs in pregnancy the greater the number of uninfected piglets born in infected litters. Transplacental infection of the porcine fetus with both field and vaccine strains of the virus may induce a spectrum of abnormalities including hypoplasia of the lungs, malformation of the pulmonary artery, micrognathia, arthrogryposis, fissures in the renal cortex, multiple septa in the gallbladder and malformations of the brain. Infection of the fetus at a critical stage of gestation (30 days) induces retardation in growth and maturation of the brain, resulting in microencephaly. The teratogenicity of the virus clearly depends on the stage of gestation. In general, the earlier the infection occurs the more severe the abnormalities are likely to be. The virus can be found in the ovaries because the blood vessels deliver peripheral macrophages to the ovaries through atretic follicles.73
One of the sequelae of transplacental hog cholera virus infection of the fetus is congenital persistent virus infection with the evolution of a runt-like syndrome during the first few months of life.52 At birth, affected piglets appear normal, although they are viremic and the viremia persists throughout life of the animals. The first evidence of clinical disease may occur at about 10 weeks of age but it may be delayed until 4 months of age. Growth retardation, anorexia and depression, conjunctivitis, dermatitis, intermittent diarrhea, and locomotor disturbance with posterior paresis occur. At necropsy, the most remarkable lesion is atrophy of the thymus gland and lesions of classical hog cholera are not present. In experimental congenital persistent hog cholera infection, the earlier the infection occurs in pregnancy the greater the number of persistent infections in piglets born alive with immunological tolerance.52 The immunological tolerance is specific to the virus because affected piglets respond to other selected antigens.
The experimental infection of pregnant goats with the hog cholera virus on days 64–84 of gestation can result in transplacental infection with the virus replicating and persisting in the fetuses for at least 40–61 days. The virus is highly pathogenic for goat fetuses and serum antibodies may be present in the pre-colostral sera of the kids.
Nearly always the detection is too late because it has been missed.74,75 The clinical signs are often non-specific but the score system suggested by the Dutch may help to suggest it.76 The differences in the four most recent German outbreaks in terms of clinical and pathological signs were minimal.77 In former times most of the European outbreaks were associated with the virulent genotype 1 of the virus but now they are types 2:1, 2:2 or 2:3,78,79 which are much less virulent and therefore produce a milder clinical course that is much more difficult to recognize over the first 14 days post-infection. In a recent set of experiments (with a strain of virus SF0277) all the pigs died but in other experiments80 some of the pigs survived.
A recent report has suggested that the occurrence of PRRS does not appear to potentiate the clinical outcome of CSF in young pigs,81 but this has been disputed.48
Simultaneous infection with Trypanosoma evansi does seem to produce a poor response to CSF vaccination.82
As a result of the recent outbreak in the Netherlands a quantitative retrospective analysis was made of the clinical signs83 which suggested that the clinical inspection was the most important part of detection but was not very specific. Moderate virulence and low virulence strains cause a mild disease that may be so mild that clinical disease is not suspected.84
Differential diagnosis should include PRRS, PDNS,85 ASF, salmonellosis, and coumarin poisoning.
Clinical signs usually appear 5–10 days after infection but incubation periods up to 35 days or more are recorded. At the beginning of an outbreak, young pigs may die peracutely without evidence of clinical signs having occurred. Acute cases are the most common. Affected pigs are depressed, do not eat, and stand in a drooped position with their tails hanging. They are disinclined to move and, when forced, do so with a swaying movement of the hindquarters. They tend to lie down and burrow into the bedding, often piled one on top of the other. Prior to the appearance of other signs, a high temperature (40.5–41.5°C; 105–107°F) is usual. In recent European outbreaks respiratory signs have not been common. Constipation followed by diarrhea and vomiting also occur. Later a diffuse purplish discoloration of the abdominal skin occurs. Small areas of necrosis are sometimes seen on the edges of the ears, on the tail and lips of the vulva. A degree of conjunctivitis is usual and in some pigs the eyelids are stuck together by dried, purulent exudate. Nervous signs often occur in the early stages of illness and include circling, incoordination, muscle tremor and convulsions. Death can be expected 5–7 days after the commencement of illness. Infection with Salmonella choleraesuis may also be potentiated by hog cholera infection and the two diseases in combination can result in high mortality.
A form of the disease in which nervous signs predominate is attributed to a variant strain of the virus. The incubation period is often shorter and the course of the disease more acute than usual. Pigs in lateral recumbency show a tetanic convulsion for 10–15 seconds followed by a clonic convulsion of 30–40 seconds. The convulsion may be accompanied by loud squealing and may occur constantly or at intervals of several hours, often being followed by a period of terminal coma. In some cases convulsions do not occur but nervous involvement is manifested by coarse tremor of the body and limb muscles. Apparent blindness, stumbling and allotriophagia have also been observed.
Low virulence strains of virus result in less severe disease syndromes.86 A chronic form occurs in field outbreaks and occasionally after serum–virus simultaneous vaccination. The incubation period is longer than normal and there is depression, anorexia, persistent mild fever, unthriftiness, the appearance of characteristic skin lesions including alopecia, dermatitis, blotching of the ears and a terminal, deep purple coloration of the abdominal skin. Pigs may apparently recover following a short period of illness but subsequently relapse and die if stressed.
Pigs infected with the low virulence strains of the virus appear more susceptible to intercurrent bacterial disease. The changeable nature of this combination is such that hog cholera should be suspected in a herd or area where there is an increase in mortality from any apparent infectious cause that either does not respond, or responds only temporarily, to therapeutic ploys that are usually effective.
Reproductive failure can be a significant feature and may occur without other clinical evidence of disease within the herd. It may occur when inadequately protected pregnant sows are exposed to virulent virus, or when susceptible pregnant sows are vaccinated with live attenuated vaccines or exposed to low-virulent field strains. Infection of the sow may result in no clinical signs other than a mild pyrexia, but it may be followed by a high incidence of abortion, low litter size, mummification, stillbirth and anomalies of piglets.52 Liveborn pigs, although carriers, may be weak or clinically normal. Persistent congenital infection is characterized by persistent viremia, continuous virus excretion and late onset of disease, with death occurring 2–11 months after birth. No antibodies to the virus are present in spite of the persistent infection; affected pigs have a normal immune response to other antigens, but do not respond to the hog cholera virus.52 Cell-mediated immunity appears to be normal. A high incidence of myoclonia congenita (congenital trembles) associated with cerebellar hypoplasia has been observed in some outbreaks where prenatal infection with hog cholera virus has occurred and this syndrome has been reproduced experimentally.52 The prevalence of any one of these manifestations appears to vary with the strain of the virus and the stage of gestation at the time of infection.52
A valuable antemortem diagnostic test is the total and differential leukocyte count. In the early stages of the disease there is a marked leukopenia, the total count falling from a normal range of 14 000–24 000 μl to 4000–9000 μl.35 This is specifically a granulocytopenia caused by a bone marrow atrophy.54 It is a result of apoptosis or necrosis, from 1–3 days post-infection probably as a result of cytokine interaction. Depletion of the lymphocyte sub-populations occurs 1–4 days before the virus can be detected by RT-PCR on serum. If a virulent form depletion is evident by 2 days.
B-lymphocytes, T-helper cells and cytotoxic T-cells are the most affected by the virus. The loss of the circulating B-lymphocytes was consistent with the failure to generate a circulating neutralizing antibody.87 Virulent strains produce a more reduction in B-lymphocytes than do mild forms.88 This can be of value in differentiation from bacterial septicemias but it should not be used as the sole method of differentiation. In the late stages of hog cholera, a leukocytosis due to secondary bacterial invasion may develop. Piglets less than 5 weeks of age normally have low leukocyte counts.
A comparison of diagnostic tests shows that the best results are detected by RT-PCR (98.9%) which is earlier than VI on blood which gives only a result of 94.5%. RT-PCR is expensive and labor intensive. The antigen-ELISA gives a later detection and the worst results.89 The leukocyte count gives the earliest pointer to CSF infection but of course does not confirm the disease.
The advent of eradication programs has resulted in the development of diagnostic tests for hog cholera. These tests must be accurate and rapid so that control measures can be rapidly instituted or lifted as required. Diagnosis by virus isolation is slow, cytopathic effect may be minimal and some strains have low infectivity and limited growth in tissue culture. This method is seldom used as a primary diagnostic method. Animal inoculation tests still provide an excellent method for the diagnosis of hog cholera and involve the challenge of susceptible and immune pigs with suspect material followed by subsequent challenge at a later date with fully virulent hog cholera virus. However, this test is time-consuming and costly and, although it is used for the final confirmatory test for the presence of hog cholera infection in various situations, it is not satisfactory for a rapid diagnostic test.
The more rapid tests rely on the detection of antigen in infected pig tissues or the detection of antibody following infection.
This technique allows the rapid detection of antigen in frozen sections of tissue or impression smears and in infected tissue cultures and these methods have been adopted as a primary test in the eradication program in the United States. Antigen can be detected up to 2 days after death and this method has been considered more reliable than the agar gel precipitation test. The method is capable of detecting virus carriers among vaccinated pigs.
The antigen-capture enzyme-linked immunosorbent assay (ELISA) can detect the virus antigens in blood and tissues from experimentally infected pigs at 4–6 days after infection with a moderate–high virulent strain (Weybridge virus) and 7–9 days after infection with a low-virulent strain (New South Wales virus).90 The technique does not require tissue culture and takes less than 36 hours for a definitive result.
A polymerase chain reaction (PCR) assay can be used to differentiate classical swine fever virus from ruminant pestiviruses.91 An international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses has been developed.92 Restriction endonuclease cleavage of PCR amplicons can distinguish between vaccine strains and European field viruses.93 The RT-PCR can also detect CSF in boar semen.94 A RT-PCR was then described.95 Rapid detection of CSF using a portable real time reverse transcriptase PCR (RT-PCR; TaqMan) has been described.96 Further modifications have been described97 so that the test can be performed in a single tube with all the ingredients. It can then be used as a pen-side test and detects virus in nasal and tonsil scrapings 2–4 days before the onset of clinical signs. A further modification of RT-PCR and ISH has been that they can be used on formalin fixed sections.98 A multiplex PCR is available to separate BVD from CSF.99
Antibody can be detected by the fluorescent antibody neutralization test, tissue culture serum neutralization test or an indirect ELISA. Serological tests are less satisfactory for detection of hog cholera in the acute phase and are of limited value in vaccinated animals. They are of value in the detection in sows of the subclinical infection of hog cholera associated with reproductive failure and for survey studies to determine the prevalence of hog cholera infection. BVDV may infect pigs, especially those in close contact with cattle, and may give false-positive serological reactions. The incidence of these false-positive reactions may be high and they pose a problem for hog cholera identification in eradication programs. The neutralizing peroxidase-linked antibody assay is a highly sensitive and specific test for hog cholera and will distinguish between pigs infected with different strains of the hog cholera virus and BVDV. The complex, trapping, blocking ELISA is sensitive, specific and reliable for screening purposes for early identification of infected herds and their elimination in an eradication program.100 A peroxidase-labeled antibody assay can be used to detect swine IgG antibodies to hog cholera and BVDVs.101 Monoclonal antibodies to pestiviruses are also available to discriminate between both viruses.102,151 A competitive ELISA using a truncated E2 recombinant protein has been described which can be used when a large number of samples are to be tested.103
When hog cholera is suspected, tissues submitted for examination should include the brain and sections of intestine and other internal organs in formalin, and pancreas, lymph node and tonsil unpreserved in sealed containers. Local regulations and requirements should be followed. The viral antigens are densely distributed in the skin and tongue of infected pigs, and biopsies of ear may be useful for diagnosis on a herd basis.104
In many cases the single most important diagnostic aid is the post-mortem examination although in the Dutch outbreaks it was thought that the contribution to the detection of CSF was limited.105,106 The reason for this is that there is tremendous individual variation. In the outbreak in the UK in 2000 there were few lesions in fetuses or in neonates and in the sows lesions were often restricted to conjunctivitis and lesions in the hepatic and splenic lymph nodes even though 15 animals in each group were examined. The only group showing more consistent lesions were the growers and in these the lesions were similar to those that are reported in the classical outbreaks.
In peracute cases there may be no gross changes at necropsy. In the more common acute form, there are many submucosal and subserosal hemorrhages but these are inconstant and to find them it may be necessary to examine several carcasses from an outbreak. The hemorrhage results from erythrodiapedesis and increased vascular permeability, probably aided by mast cell degranulation.107 The hemorrhages are most noticeable under the capsule of the kidney, about the ileocecal valve, in the cortical sinuses of the lymph nodes and in the bladder and larynx. The hemorrhages are usually petechial and rarely ecchymotic. The lymph nodes are enlarged and the spleen may contain marginal infarcts. Infarction in the mucosa of the gallbladder is a common but not constant finding and appears to be an almost pathognomonic lesion. There is congestion of the liver and bone marrow and often of the lungs. Circular, raised button ulcers in the colonic mucosa are usual but cannot be distinguished from those of salmonellosis. Although these gross necropsy findings are fairly typical in cases of hog cholera, they cannot be considered as diagnostic unless accompanied by the clinical and epizootological evidence of the disease. They can occur in other diseases, particularly salmonellosis. In a recent study77 found that the lymph nodes had the highest score for lesions and that the least lesions were found in the spleen and tonsil because infection of these organs was also rare. The most common lesions were also in the lymph nodes, around the ileo-caeco-colic junction and around the blood vessels of the brain. Atypical bronchiolar cilia have been reported.108
There are characteristic microscopic lesions of a non-suppurative encephalitis in most cases and a presumptive diagnosis of hog cholera can be made if they are present. It is thought that the most common lesion in chronic CSF is the mononuclear cell cuff in the CNS. Here ISH is capable of detecting viral nucleic acid even when viral antigen is not detected.94 Histologically, the main site of tissue injury is the reticuloendothelial system. There is always a progressive lymphoid depletion and mucosal necrosis. The depletion is probably caused by apoptosis but not by direct apoptosis. Atrophy of the thymic cortex and loss of thymocytes is also a feature and may be related to synthesis of the cytokines, TNFα and IL-1α in particular109 which may increase the apoptosis of the thymocytes.
Fibrinoid necrosis of the tunica media combined with hydropic degeneration and proliferation of the vascular endothelium causes occlusion of blood vessels. The more virulent ‘neurotropic’ strains produce lesions of a similar nature but greater severity.
In the intestinal tract mucosa there are large, usually infected macrophages.110 The GALT areas are lymphocyte depleted usually because of massive lymphocyte apoptosis particularly in the B-cell areas. These changes are possibly due to the large amounts of TNFα and IL-1α released from the infected macrophages.111 They also showed that the macrophages in the splenic marginal zone were amongst the first cells to be infected. The infection, mobilization, and apoptosis of splenic macrophages plays a very important role in the course of the infection through cytokine release. An unusual manifestation of CSF infection is the onset of metaphyseal bone formation caused by the partly thrombosed vessels in the bone with strong CSF viral specific fluorescence.112
Histology, showed swelling and vacuolation of megakaryocytes in the bone marrow 2 days after infection and they were necrotic 4 days after infection.113 Severe swelling and necrosis of endothelial cells in the vascular endothelium were observed 3 days after infection. It was concluded that the thrombocytopenia due to direct viral damage to MKC and endothelial damage can cause hemorrhagic diathesis whereas coagulation disorders are not involved in early stages of the disease.114
In the chronic form of the disease, ulceration of the mucosa of the large intestine is usual. Secondary pneumonia and enteritis commonly accompany the primary lesions of hog cholera.
Infection of the fetus produces a persistent immunologically tolerant non-cytolytic infection, often with little evidence of cell necrosis or inflammatory reaction to suggest the presence of a virus. Aborted fetuses show non-diagnostic changes of petechial hemorrhage and ascites. Malformations such as microcephaly, cerebellar hypoplasia, pulmonary hypogenesis and joint deformity appear due to inhibition of cell division and function in these areas. Antibody is not detected in fetal blood when infection occurs early in fetal life. In pigs showing signs of myoclonia congenita, cerebellar hypoplasia is highly suggestive of hog cholera infection.
An immune complex glomerulonephritis has been described115 in which there is macrophage infiltration of the mesangium with immune complex deposits of IgM, IgG, and Clq in mesangial, subepithelial and subendothelial areas from 10 days post-infection and by 14 days neutrophils had also congregated.116
This is a disease of major economic importance and confirmation of the diagnosis is usually performed in specialized governmental laboratories. Virus isolation and fluorescent antibody tests104 are most commonly used but other techniques, including immunoperoxidase staining of cryostat sections.92 The demonstration of viral antigen in the crypts of the tonsils, tubular epithelial cells of the kidney, bronchiolar mucosal gland cells and the pancreatic epithelial cells has been shown to be possible even after 18 years in formalin.117
Fresh tonsil can be used for polyclonal direct fluorescent antibody which also detects BVD and BDV and can then use additional tests. The sensitivity of this test was shown to be only 78% so to give a 99% chance of infection being detected you had to postmortem five animals.118
• Histology – formalin-fixed brain, spleen, lymph nodes, colon, cecum, ileum, kidney, tonsil, skin, tongue (LM). Tissue sections can also be used for ISH and IHC115
• Virology – lymph nodes, tonsil, spleen, distal ileum, skin, tongue, brain (FAT, ISO, IHC, PCR). Heparinized blood.
A positive diagnosis of hog cholera is difficult to make without laboratory confirmation. This is particularly true of the chronic, less dramatic forms of the disease. A highly infectious, fatal disease of pigs with a course of 5–7 d in a group of unvaccinated animals should arouse suspicion of hog cholera, especially if there are no signs indicative of localization in particular organs. Nervous signs are probably the one exception. The gross necropsy findings are also non-specific and reliance must be placed on the leukopenia in the early stages and the non-suppurative encephalitis visible on histological examination. Both of these bacterial infection, particularly salmonellosis, is present.
The major diseases which resemble hog cholera include:
• Salmonellosis usually accompanied by enteritis and dyspnea
• Erysipelas in which there are characteristic diamond skin lesions, and the subserous hemorrhages are likely to be ecchymotic rather than petechial;
• Pasteurellosis in which respiratory signs predominate and lesions of pleuropneumonia at necropsy are characteristic.
Epidemiological considerations and hematological and bacteriological examination will usually differentiate these conditions.
Other encephalitides, particularly viral encephalomyelitis and salmonellosis cause similar nervous signs.
African swine fever apart from its greater severity, is almost impossible to differentiate from hog cholera without laboratory testing.
Hyperimmune serum is the only available treatment and may be of value in the very early stages of the illness if given in doses of 50–150 mL. It has more general use in the protection of in-contact animals. A concentrated serum permitting the use of much smaller doses is now available.
The methods used in the control include eradication and control by vaccination. Both modeling and real time prediction have been described.119 In areas where effective barriers to reintroduction of the disease can be established, eradication of the disease by slaughter methods is feasible and usually desirable. In contrast, in areas where the structure and economics of the pig industry require considerable within-country and across-border movement of pigs, it may not be practical or economically feasible to institute a slaughter eradication program. The establishment of a highly susceptible population in a high-risk area is unwise. If repeated breakdowns occur, the restriction of movement of pigs within the quarantine areas creates considerable managemental problems for pig owners and they may, as a result, eventually become non-cooperative in the program. In these areas, control and possibly even eradication by vaccination is the approach of choice and this method is used in some countries like the Phillipines.18 The Commission for the European Communities has declared its policy, supported by appropriate community legislation, to eliminate hog cholera without vaccination.17 A full discussion of the possibility of using vaccination in the future has been outlined.120 A computerized framework for the risk assessment for CSF has been produced.150 In Germany there are big risks with regard to the import of pigs, wild boars and the import of pig meat.121 A retrospective spatial and statistic simulation to compare two vaccination techniques with the non-vaccination scenario in the Dutch 1997/1998 CSF outbreak showed that both emergency vaccination techniques would hardly have been more efficient.122 General procedures are described first followed by a description of the immunizing products available.
Modelling for the control of CSF in such areas has been described B.123
In areas where the disease does not normally occur, eradication by slaughter of all in-contact and infected pigs is possible and recommended. The pigs are slaughtered and disposed of, preferably by burning. All herds in the area should be quarantined and no movement of pigs permitted unless for immediate slaughter. In areas with high pig densities, control strategies depend on highly effective identification and recording systems which provide information on herd inventories and animal movements so that herd epidemics can be traced back to their origin.17 Recent experiences with epidemics of swine fever in Belgium and the Netherlands found that, with the current ear tag with manual recording and use of documents system, most epidemics could not be traced back to their origin.17 The tracing and removal of carrier herds prevents these herds from becoming infectious and prevents the spread of disease at an early stage.
All vehicles used for the transport of pigs, all pens and premises and utensils must be disinfected with strong chemical disinfectant such as 5% cresylic acid. Contaminated clothing should be boiled. Entry to and departure from infected premises must be carefully controlled to avoid spread of the disease on footwear, clothes and automobile tires. Legislation prohibiting the feeding of garbage or commanding the boiling of all garbage before feeding must be enforced. This eradication procedure has controlled outbreaks which have occurred in Canada and Australia and has served to maintain these countries as free from the disease.
One of the major problems in Europe is the wild life reservoir in the wild boar population. In areas where there is little risk there are few positive animals but where there is a high risk then many animals may be positive. In Switzerland 179 of 528 boars in a risk area were positive.124 The oral vaccination of wild boars was described in Germany125,126 has no risk for the establishment of a persistent wild boar CSF infection.127 However it was shown that more than 50% of the wild boar did not feed on the vaccination baits and therefore did not become immune.125 There is evidence from wild boar studies in Italy that the level of infection in the free population gradually reduces in any case.129 Where you have wild pigs with maternal antibodies when they contract live CSF virus they have transient clinical signs but the disease is not lethal. Infected wild boars could therefore play a very big part in the transmission of a natural outbreak.130 The vaccination studies in wild boar were reported recently and showed that after the 5th vaccination there was no viremia, no virus excretion, and no post mortem virus recovery.131 Oral vaccination of wild boar usually reduces the presence of CSF but only a low rate of wild boar (30–35%) become seropositive.132
In endemic areas, control is mostly a problem of selecting the best vaccine and using it judiciously. In Asia almost all the control is vested in the use of vaccines and their proper use. Most problems are caused by policy failures, or changes in demographics whereas most of a vaccination policy should be determined by the epidemiology of the disease. Much can also be done to keep the incidence of the disease low by the education of farmers whose cooperation can be best assured by a demonstration that eradication is both desirable and practicable. Once farmers are motivated to act, the greatest stumbling block to control, failure to notify outbreaks, is eliminated. Education of the farmer should emphasize the highly contagious nature of the disease and the ease with which it can be spread by the feeding of uncooked garbage and the purchase and sale of infected or in-contact pigs. The common practice of sending pigs to market as soon as illness appears in a group is one of the major methods by which hog cholera is spread.
There are two sorts of vaccine:133
1. The first group is the classical live group containing attenuated virus and these are to be preferred. Live, virulent virus vaccines produce a solid immunity within just a few days and give lifelong protection but are capable of introducing the infection and of actually causing the disease when vaccination ‘breaks’ occur. The reaction to live virus vaccine may be severe and the susceptibility of pigs to other diseases may be increased. Eradication of the disease is impossible while the use of this type of vaccine is permitted.
2. There is a recently developed second group of live vaccines aimed as marker vaccines134 based on the E2 protein135 but these are still undergoing development. There appears to be no complete protection against congenital infection, they do reduce transmission of virus136 and they seem to only last about 1 year.137 They do have the potential to allow tests to be used to differentiate between naturally infected and vaccinated animals.138 They may also fail in the face of natural infection.139-141 Recent further developments of these marker vaccines77 possibly include a chimeric vaccine where one of the genes has been replaced by a BVD gene and a second vaccine where a DNA vaccine expresses the E2 protein after entering the host cell and others with E2 peptides.142-145
When an outbreak occurs in a herd, the immediate need is to prevent infection from spreading further. This can be best achieved by removing the source of infection and increasing the resistance of in-contact animals by the administration of hyperimmune serum or one of the available vaccines. Removal of the source of infection necessitates:
• Isolation of infected animals. This was highlighted in the recent Dutch outbreak146
• Suitable hygienic precautions to prevent the spread of infections on boots, clothing and utensils
• Disposal of carcasses by burning
• Disinfection of pens. The pens should be scraped, hosed and sprayed with 5% cresylic acid solution or another suitable disinfectant. The choice of serum or vaccine may depend on local legislation and will depend upon circumstances. Pigs in the affected pen should receive serum (20–75 mL depending on size) and pigs in unaffected pens should be vaccinated. Pigs receiving serum only will require active vaccination at a later date if a strong immunity is to be achieved. Routine vaccination of all pigs is desirable.
The elimination of hog cholera from a country where it is well established presents a formidable problem. Before the final stage of eradication can be attempted, the incidence of the disease must be reduced to a low level by widespread use of vaccination and enforcement of garbage-cooking regulations.
One of the most important problems encountered in eradication programs is the clinically normal ‘carrier’ animal and steps need to be taken to avoid the sale of all pigs from infected premises. A procedure which has been particularly effective in the control of this and other diseases of pigs is the complete prohibition of all community sales of feeder pigs. There are obvious political difficulties in such a prohibition, but despite their usefulness as marketing agencies, community sales continue to be a major source of swine infections. When the occurrence of virulent hog cholera has been eliminated, further necessary steps include the prohibition of use of any vaccine and serological studies to detect low virulence carrier states.
The eradication of swine fever in the United Kingdom in 1986 was an important achievement. Control was radical in that all herds in which the disease was diagnosed were slaughtered and all carcasses burned or buried to avoid missing atypical cases and recurrence through the swill cycle. The two focal points which became apparent were the need to avoid vaccination and the need to diagnose accurately. Vaccination was not permitted because it was not completely effective, produced ‘carriers’, and encouraged the development of mild and chronic forms of the disease. The need to diagnose accurately led to changes in diagnostic procedure as the campaign progressed. As the proportion of classical epidemics declined, there was increasing dependence on serological and antigen-detection tests. The program in the United States, which currently appears complete, is an equivalent achievement.
Very few pigs possess natural immunity to hog cholera and, until the introduction of the serum–virus method of vaccination, an outbreak of the disease in a herd meant virtually that the herd would be eliminated. The situation changed rapidly thereafter and it can be safely claimed that the development of the swine industry in the United States would have been impossible without the protection which the serum and virus provided. On the other hand, the dangers inherent in the use of fully virulent or partially avirulent virus do not recommend their use and have led to a continuing search for safe methods of immunization. The ideal vaccine should retain strong immunogenicity but should be completely avirulent, even for pregnant sows, the fetus and young or stressed pigs. It should be stable in the degree of attenuation and should not persist in the vaccinate nor transmit from the vaccinate to in-contact pigs. Killed vaccines are safe and do not directly spread virus, but in general, they engender only a limited immunity. Live vaccines provide a longer lasting immunity but frequently have not met the criteria listed above.
The serum–virus vaccination produces an immediate, solid and lasting immunity when properly administered to healthy swine. The virus, produced by collecting blood 6–7 days after artificial infection, is injected SC in 2 mL doses followed immediately by serum in doses graduated to the size of the pigs and varying from 20 mL for suckling pigs to 75 mL for adults. Overdosing with serum will not prevent the development of immunity. Vaccination is performed at any age after 4 weeks. Because of the availability of safer vaccines this method is not recommended.
Attenuated vaccines include tissue culture vaccines attenuated by repeated passage through tissue culture of porcine or other origin, lapinized vaccines produced by repeated rabbit passage, and vaccines from mutant strains. Many of the early vaccines of this type were not stable and could cause disease when not used in conjunction with serum. Furthermore, transmission to in-contact pigs, especially with porcine origin vaccines, and fetal disease following vaccination of pregnant sows have been problems. Attenuated vaccines are in wide use in Europe and Asia and include the Chinese or LPC and GPE strains. Recently, a safe and efficacious CSF marker vaccine based on the E2 protein (major immunogen) of the virus has been developed. This protein has neutralizing antibodies and it is also conserved. It can be used where there is an endemic problem and also after the outbreak.147 It also prevents or reduces drastically the problem of trans-placental infection.148 Experimental, non-transmissible marker vaccines have also been developed.149 Highly passaged preparations are antigenically stable and show no evidence of reversion. They produce a very limited viremia, or none, and no leukopenia or clinical illness. Protection is evident within 5–10 days of vaccination. Piglets from non-immune sows can be vaccinated within the first 2 weeks of life. Because the presence of maternal immunity can interfere with effective immunization, the vaccination of piglets from immune sows should be delayed until at least the second month. The French Thiverval strain is a cold mutant strain which has lost its virulence but retained good immunogenicity. Vaccination of piglets even with ten times the regular dose produced no clinical illness and virtually no viremia. A single IM vaccination will produce resistance to challenge by 5–10 days and immunity persists for 3 years. Colostral immunity will protect piglets for periods up to 2 months after birth. When given to pregnant sows, even the highly attenuated strains have the ability to cross the placenta and produce fetal infection even though no clinical evidence of this may be manifest. Consequently it is recommended that replacement gilts be vaccinated at least 2 weeks prior to mating and that recently vaccinated animals be kept separate from susceptible pregnant sows.
In the Netherlands, the control of swine fever has relied on a slaughter policy on affected farms plus an emergency vaccination program in which all pigs over 2 weeks of age in areas of risk are vaccinated. The mass vaccination program is followed by supplementary vaccination of pigs at 7–9 weeks of age and revaccination of breeding gilts when they reach when 6–7 months of age. The serological response of piglets born from vaccinated sows is best at 9–10 weeks of age rather than at 5–6 weeks of age. A vaccine-induced neutralizing antibody titer of <32 is adequate to provide protection against clinical disease and to prevent virus transmission.
These are usually prepared from the blood or tissues of infected pigs. Crystal violet vaccine has been the one most widely used of this type and was used in the United Kingdom prior to eradication but never gained full acceptance in the United States. It is completely safe but its immunogenicity is poor. Immunity does not develop until 12 days after vaccination. Its duration is short and booster injections are required for maintenance. Vaccinated sows may still develop fetal infection when exposed to virulent virus and there is a danger that the use of this vaccine in enzootic areas may in this way induce virus carriers. The production of immune antibodies to the blood components of the vaccine may result in the occurrence of isoimmune hemolytic anemia in some breeds. For these reasons inactivated vaccines are not in common use.
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This is an OIE List A disease. It is indistinguishable in the field from classical swine fever as both are hemorrhagic diatheses and it is just as contagious. However, it is associated with a totally different virus.
Disease of major threat to pig-producing countries. Occurs in Africa, western European countries, Caribbean countries. High morbidity, high case-fatality rate in classic form; low virulence form less fatal. In Africa, transmitted by argasid tick from wild pigs to domestic pigs. In Europe transmitted by direct contact with infected pigs
Antibodies in colostrum of recovered sows provide passive protection to piglets
High fever, purplish skin, depression, anorexia, huddling, disinclination to move, weakness, incoordination, nasal and ocular discharges, diarrhea, vomiting, abortions, death in a few days. Historically, highly virulent forms; recent decades subacute and chronic forms common with fever, depression, lethargic, recover in few weeks but remain persistently infected; chronic cases are intermittently pyrexic and become emaciated with soft edematous swelling over joints and mandible
It is associated with a DNA virus which is the sole member of the Asfariviridae and as such is the only known DNA arbovirus.1 It is a large icosahedral virus that contains a linear, double-stranded DNA genome (170–190 kbp). The viral genome may encode for 165 genes2 and encodes for approximately 113 viral proteins, most with an as yet unknown function.3 Morphologically, it is similar to the iridoviruses but resembles the pox viruses in genome construction and gene expression. There are different forms from highly lethal to sub-clinical with different field strains and tissue culture adapted strains.4 These are recognized by restriction fragment length polymorphism (RFLP) and protein p72 recognizes all viral groups. Partial p72 gene characterization allows genotyping of field strains.5
African swine fever is indigenous to the African continent where it affects wild pigs. These include wart hogs, bush pigs and escaped (feral) forest hogs which act as reservoirs of the virus which cycles between the pigs and the ticks. Wild pigs in some areas are free of infection and consequently the disease is not endemic in all areas. It was always considered to be a disease of Sub-Saharan Africa but over the years has reached new areas. The occurrence has recently been reviewed.6 These include Mozambique in 1994, Kenya in 1994, Ivory Coast in 1996,7 Benin in 1997 and also Togo and Nigeria in 2001.8 The Kenyan outbreak seemed to be maintained in the domestic pigs without sylvatic hosts.9 The Nigerian strain was 92–97% homologous to the strains from Uganda, Dominican Republic, and Spain. The serious worry was the appearance of the virus in Madagascar in 1998.10 Although studies showed only a seropositivity of 5.3%11 Infection of wild pigs produces no clinical disease but with virulent strains, infection in the domestic pig is almost always fatal. Since its recognition, occurrence of the disease in South Africa has been cyclical with periods of 10–12 years of clinical disease and then an absence of disease. Until 1957, African Swine Fever (ASF) had not occurred outside the African continent. To the rest of the world it represented the most formidable of the exotic diseases of swine, a disease which had to be kept within its existing boundaries at all costs. However, ASF spread from Africa, appearing in the domestic pig population in Portugal in 1957 and Spain in 1960; and resulted in the death or slaughter of thousands of pigs. Subsequently the disease appeared in France and then Italy and in 1971 occurred in Cuba. It was successfully eradicated from these latter three countries by extensive slaughter and quarantine programs.
The exception in Europe is Sardinia where the disease is endemic in the Central Highlands although it has decreased from Oct 1994 to March 1996. In a survey in 1998, 45 of 82 municipalities in the province of Nuoro in Sardinia were found to have ASF. The principal reasons being the extensive pig farming and the occurrence of wild boar.12 The partial confinement farms have less seropositivity than the free-range farms13 and those in total confinement have only 20% of the level of the free-range farms. In 1978, outbreaks occurred in Malta,14 Brazil, Dominican Republic and Haiti. In Malta, the disease resulted in the death or slaughter of the entire population of 80000 pigs within 12 months of the diagnosis.14 This is one of the few examples where a country had to slaughter an entire species of a domestic animal in order to eliminate a disease. An outbreak occurred in Belgium in March 1985. The source of infection was thought to be pork imported from Spain, which was fed to only one boar. Once the official diagnosis was made, all animals on affected farms were slaughtered. Animals which had direct commercial contact with infected herds were also slaughtered and the disease was declared eradicated in September 1985. In Spain, the disease had been present since 1960 but the implementation of regulations for eradication adopted in 1985 made it possible to divide the country into an African swine fever-free region and an infected region.15 Since 1995 Spain and Portugal have been declared free from the disease although there was an isolated outbreak in Portugal in 1995. This has resulted in a marked change in the distribution and incidence of the disease.
Only pigs are affected; domestic pigs of all ages and breeds are highly susceptible, but the virus can be passed in tissue cultures of rabbits, goats and embryonated hen eggs.
Until recently, the occurrence of the disease in Africa was limited to explosive outbreaks in European pigs which came in contact with indigenous African pigs. These outbreaks tended to be self-limiting because all pigs in affected herds died or were destroyed, but after a number of years the disease became enzootic in domestic herds. Surveys of the disease in countries like Malawi illustrate the changing behavior of the disease over a period of years. The virus which was introduced to Europe in 1957, was capable of persisting in European pigs and after a period of several years in which the disease was epizootic, a change to an enzootic character occurred. The outbreak in Cuba was of a comparatively virulent form.
When the disease occurred in the Caribbean region, it posed a major threat to the large swine industry of the United States principally because of the possible spread of the virus to the feral swine population in Florida.16 The feral swine population in Florida is the largest in the United States and is of major recreational and economic importance to hunters, trappers, taxidermists and dealers who sell feral swine to hunting clubs. The feral swine in Florida are descendants of domestic swine which were allowed to run wild. Experimental inoculation of these pigs with virulent isolates of the virus will cause fatal disease.
Early in the history of African swine fever, the morbidity rate could be as high as 100% and the case–fatality rate was also often over 90%. However, a decrease in the virulence of the virus occurs with time in enzootic areas, and the case–fatality rate may now be as low as 2–3%.17
In Africa, the method of transmission of the disease from the reservoir in wild pigs to the domestic pig has been the subject of considerable interest. Infection is primarily transmitted to domestic pigs via the argasid tick Ornithodoros moubata. The viremic wart hog is a source of infection for the ticks. The virus can be maintained in wart hog-associated argasid ticks by a trans-stadial, transovarial and sexual (male to female, but usually not vice versa) transmission mechanism. It needs to replicate in the mid-gut epithelium of the tick for successful ASF infection of the tick.18 The tick is relatively restricted in its habitat and if contact between domestic pigs and wild pigs and their burrows is prevented, transmission can be prevented.14 The virus can be maintained in these ticks for long periods in the absence of fresh sources of infection, with a low level of viremia lasting a long time. The young wart hogs in the burrows are infected early on, so that they act as a reservoir as well as vectors of infection. Sporadic outbreaks may thus occur in endemic areas when the virus spreads from infected ticks or wart hogs to domestic pigs. In some areas where infected wart hogs are common but where O. moubata is apparently absent, O. savignyi may be a natural field vector of the virus. It is also found in O. porcinus porcinus.19,20 The ASF virus replicates to a high titer in the developing cells of the egg of the tick.21 Ticks infected with ASF virus also have a higher mortality than uninfected ticks.22
The long-held belief that the source of the virus in primary epidemics of African swine fever in southern and eastern Africa is the carrier, wild pig, is not tenable. It is postulated that infected ticks are transported to the vicinity of domestic pigs either by wart hogs or on the carcasses of wart hogs.
In Africa, the virus is maintained primarily by a cycle of infection between wart hogs and soft ticks (Ornithodoros moubata).14 The virus does not have an apparent effect on either wart hogs or ticks and it is only when infection of domestic pigs occurs that the virus produces disease. Indeed most wart hogs are aviremic but seropositive. The tick has a wide distribution in Africa south of the Sahara, and its main habitat is in burrows which are inhabited by the wart hog. There is a good correlation between antibodies in wart hogs and the presence of ticks. Newborn wart hogs can become infected soon after birth if bitten by infected ticks and the consequent viremia would be high enough to infect previously uninfected ticks feeding on them. It is also found in the bush pig (Potamochoerus porcus)23,24 which following infection may be viremic for 35–91 days, and these also transmit the infection to ticks.
In Spain and Portugal, the methods of spread are contact between neighboring farms and the introduction of infected pigs either during the incubation period or as persistently infected virus carriers. During the last 20 years, an increasing number of outbreaks occurred in which clinical disease was not readily recognized. The mortality rates decreased and a wide range of clinical disease occurred, ranging from acute to chronic and including apparent recovery to normal health. The major consequence of the emergence of these less virulent forms of the virus was the development of persistently viremic carriers and a large population of pigs with inapparent infection. The African swine fever virus may persist in the pig population by persistent infection in recovered pigs for several months, during which time the virus must be reactivated before transmission can occur. The virus can also persist by reinfection of recovered pigs in which virus replicates without producing clinical disease and transmission occurs by excretion and by infected blood and tissues. Wild boars in Spain carry the virus without clinical signs.25
The European vector of the virus is the soft tick O. erraticus.14 It can maintain and transmit the virus for at least 300 days. In various areas of Spain, O. erraticus was found in 42–64% of the pens occupied by pigs.26 Following the outbreak of the disease in Spain, abandonment of these pig pens has resulted in the elimination of most soft ticks infected with the virus.26 The adults and large nymphs can survive for about 5 years or longer in the soil of pig pens when animals occasionally enter them. There is a relationship between the persistence of the disease and the distribution of the tick in Spain.27 Hungry tick populations may transmit the virus when feeding in the winter but populations that have continuous access to pigs do not feed until the pig pens reach a temperature of 13–15°C. The development from larva to adults takes 2–3 years. In a recent studied on the ticks (O. erraticus) from farms in Southern Portugal two types of ASF were isolated.28 One produced the acute, 100% fatal disease, and the other just a low viremia in pigs.
In Sardinia, the major factors involved in the spread of the disease are related to the:
• Mountainous terrain in which pigs may range freely in previously infected areas
• Movement of pigs which may survive infection and mingle with other herds
• Introduction of infected pigs from unknown sources into healthy herds because of the uncontrolled movement of pigs
• Feeding of waste food containing meat from infected pigs.14
The virus has been experimentally transmitted to healthy swine by O. coriaceus, an argasid tick indigenous to the United States. The potential arthropod vectors of the virus in North America and the Caribbean Basin has been examined. Most Ornithodoros spp. of ticks that will feed on pigs may be capable of acting as vectors of the virus, and the possible existence of potential vectors among the other blood-sucking arthropods should not be ignored. The soft tick O. (Alectorobius) puertoricensis found on the Caribbean Island of Hispaniola (Haiti and Dominican Republic) where African swine fever was endemic from 1978 to 1984, was experimentally able to transmit the virus from infected to susceptible pigs.29 The O. coriaceus tick is able to harbor and transmit the virus for more than 440 days, passing it trans-stadially from the first nymphal stage to the adult, sustaining it through at least four molts. O. puertoricensis has all of the prerequisites for becoming a true biological vector and reservoir of the virus.
Once established in domestic pigs the disease can spread rapidly. Virus is present in high titer in nasopharyngeal excretions at the onset of clinical signs and is present in all organs and excretions in acutely sick pigs. In experimentally inoculated domestic pigs, the virus is present in substantial amounts in secretions and excretions of acutely infected pigs for only 7–10 days after the onset of fever and is present in the greatest amount in the feces. The virus can persist in the blood of some recovered pigs for 8 weeks and in the lymphoid tissues for 12 weeks. Feces are the environmental contaminant most likely to spread the infection, but blood is also highly infective and transmission could occur by contamination of wounds created by fighting. Infection occurs via oral and nasal routes and with the short incubation period once the disease is established in a herd, it spreads rapidly by direct contact. Infection amongst domestic pigs can also reputedly be spread by:
• Indirect contact by infected pens
• Ingestion of contaminated feed and water
• Feeding uncooked garbage containing infected pig material. Transmission via the hog louse Haematopinus suis is also probable. An important source of infection is the recovered pig which may remain persistently infected and a carrier indefinitely. Pigs which have recovered from the western hemisphere isolates (Brazilian and Dominican Republic) may be persistently infected and are resistant to experimental challenge.
The African swine fever virus is a multiclonal population of viruses in which all combinations of at least four markers (hemabsorption, virulence, plaque size, and antigenicity) are found. This may explain the epidemiological observation that when the disease was confined to Africa and the Iberian peninsula in the early 1960s, the viruses isolated were highly virulent to swine, but in subsequent years mortality decreased and subacute and chronic infection became more common. Experimentally, moderately virulent African swine fever virus obtained from the Dominican Republic, when inoculated into pigs, results in an acute febrile illness along with viremia and a transient neutrophilia from which the pigs recover. The Malta 78 isolate of the virus experimentally produces a clinical syndrome similar to the African isolates of the virus.
A huge amount of research is continuing apace into the genes and the proteins produced from the expression of these genes but these are really beyond the scope of this text.30 However, recent studies of ASF have suggested that the virulence may depend on their ability to regulate the expression of macrophage derived cytokines which in turn regulate Th1 and Th2 responses and control the host protective responses.31 The less virulent cultures of ASF with macrophages produce more TNFalpha, IL-6, IL-12, and IL-15, i.e. virulent strains inhibit their production. It also affects chemotactic responses and phagocytic capacity32,33 at the same time as a reduction in the release of toxic oxygen radicals.
The virus is highly resistant to putrefaction, heat (it will survive 2 h at 56°C and dryness and survives in chilled carcasses for up to 6 months17 and at 4°C for 2 years. Probably 0.5–0.66 of all the genes of ASF are not connected with virus replication but are important for viral transmission and survival in the host.34
Antibodies against the African swine fever virus occur in the colostrum of sows previously infected with the virus and are transferred passively to nursing pigs. Experimentally, passively transferred virus-specific immunoglobulins alone will protect swine against lethal infection with a highly virulent homologous strain of the virus.35 The antibody-mediated protective effect is also an early event which effectively delays disease onset. The construction of blocking antibodies by some of the viral proteins probably prevents the complete neutralization of the virus by antibodies.36
Pigs infected with virulent or attenuated virus may recover and resist challenge exposure with virulent homologous and, under certain conditions, heterologous viruses. Although pigs develop antibodies which are detectable by different tests, virus-neutralizing antibodies have only recently been demonstrated against viral protein p72.37 However it has recently been suggested that p30, p54, p72, and p22 proteins are not associated with neutralizing antibodies.38 The sera from pigs which have been infected and are resistant will inhibit virus replication but the nature of the inhibition is not understood. Neutralization of virulent virus isolates in both Vero cell cultures and swine macrophages using swine immune sera has been demonstrated.39 Experimental exposure of pigs to a low-virulent field isolate of the virus results in a range of virus-induced specific cellular responses.
The virus induces strong in vitro blastogenesis of primed blood mononuclear cells, when less virulent, but live virus isolates are used. Pigs recovering from an acute infection with the virus have significant levels of virus-specific cytotoxic T-lymphocytes after in vitro stimulation. Viral protein p36 induces a helper T-cell response in mice.40 Resistance to infection appears to be related to the level of antibody dependent cell mediated cytotoxicity. Virus-specific blastogenic and cytotoxic T-cells are prime candidates for the cells inducing and conferring protective immunity against challenge with the virus suggesting that cellular based mechanisms are highly important.41,42
The virus invades through the tonsils and respiratory tract and replicates in the lymphoid tissues of the nasopharynx prior to the occurrence of a generalized viremia, which can occur within 48–72 hours of infection.
Infectivity and contact transmission develops at this time and continues for at least 7 d. Pigs inoculated with field isolates of the virus from the western hemisphere develop thrombocytopenia with a characteristic pattern. Infected pigs become thrombocytopenic over a 48-hour period after 3–4 days of illness. After several days of thrombocytopenia, the platelet count returns to baseline level even with a continuing viremia. Experimentally, the virus causes hematopoiesis in bone marrow which coincides with macrophage activation and bone marrow function is not impaired.43 Membrane proteins on the surface of permissive cells act as receptors for ASF and specific interactions take place at this site.44
The effects of ASF are primarily hemorrhages and apoptosis.45,46 A new protein (p54) encoded by the virus has just been shown to be the first that directly induces apoptosis.47 The disease is characterized by apoptosis with abundant lymphocyte particularly B-cell death.24 Both T and B-cells, particularly in the spleen, are affected as early as 3 days after infection. The apoptosis being induced by cytokines or apoptotic mediators released from ASF infected macrophages. In all probability there is an intra-cellular pathway triggered at the same time as the process of virus encoding.48 It is probable that the inducers of apoptosis are balanced by the inhibitors of apoptosis.49
Tissue necrosis and generalized endothelial cell infection are not features of the disease caused by isolates of moderate virulence.
The virus causes hemorrhages through its effect on hemostatic mechanisms50 by affecting vascular endothelium.46 After about 4–5 days the vascular damage extends to the basement membranes and death ensues usually because of the serious edema and hemorrhage. The mechanisms related to hemorrhage consist of the:
1. Activation and extensive destruction of monoctyes and macrophages. Serum TNFα51 and IL-1β increase in the serum.52 The lymphocytes also appear to have decreased activity.53 Apoptosis of thymocytes has been reported54
2. Disseminated intravascular coagulation
3. Infection and necrosis of megakaryocytes. Many apoptotic and also pyknotic and karryorhetic megakaryocytes55 can be seen which are induced either by cytokine damage or peripheral destruction of platelets.56 Between 0.2–9.5% of cells may be affected. Early in the infection there is prolongation of coagulation times due to inhibition of fibrin formation and later thrombocytopenia develops. The thrombocytopenia and coagulation defects lead to the development of:
All clinical forms of the disease are characterized by extensive hemorrhages at necropsy and it is this feature which often establishes a presumptive diagnosis in the field. A highly virulent virus produces renal hemorrhages as a result of intense endothelial injury, facilitated by phagoctyic activity.57 With strains of moderate virulence, hemorrhage is a consequence of an increase in vascular permeability with diapedesis of erythrocytes.57 Activation of platelets by the virus may also contribute to increased permeability.
The virus mainly infects cells of the mononuclear phagocyte system and also impairs lymphocyte function. Pulmonary intravascular macrophages demonstrate intense TNFα and IL-1α activity which coincides with the pulmonary edema, neutrophil sequestration and fibrin microthrombi.58 The lymphopenia which is so characteristic of the disease is due to a significant increase in lymphoctye death by apoptosis (programmed cell death).59 In the experimental disease, there is marked apoptosis of lymph-node lymphocytes and this occurs in both compartments of cortical tissue, but is more intense in diffuse lymphoid tissue (T area). The peripheral lymphopenia is associated with T-lymphocyte depletion. There is no evidence of virus replication in lymphocytes in the lymph nodes but there is a high rate of viral replication in macrophages in diffuse lymphoid tissue compared to the low rate in lymphoid follicles.60 In summary, there is lymphoid tissue impairment and programmed cell death of a high percentage of lymphoid and monocyte/macrophage cell populations. This accounts for the lymphopenia and the state of immunodeficiency. There are also a variety of proteins encoded by the virus that are apoptosis inhibiting proteins.61 Experimentally, the virus also causes activation and degranulation of platelets from day 3 after inoculation onwards, coinciding with activation of the mononuclear phagocyte system and virus replication in monocyte/macrophages.62 Virions of the virus also appear in the platelets, which suggests that platelets assist in disseminating the virus within the body, especially in subacute infections. Probably 95% of the infectivity of blood is in the form of virus adsorbed to the red blood cells.
The virus can cross the placenta, replicate in fetal tissues and cause abortion. However, the pregnancy failure is probably the result of the effects of the virus infection on the dam more than from direct viral damage to the placenta or fetus.63
In the acute form of the disease the animals die in an acute state of shock characterized by a disseminated intravascular coagulation with multiple hemorrhages in all tissues.64 The incubation period after contact exposure varies from 5–15 days. A high fever (40.5°C; 105°F); appears abruptly and persists, without other apparent signs, for about 4 days. The fever then subsides and the pigs show marked cyanotic blotching of the skin, depression, anorexia, huddling together, disinclination to move, weakness and incoordination. Extreme of the hindquarters with difficulty in walking is an early and characteristic signs. Coordination remains in the front legs and affected pigs may walk on them, dragging the hind legs. Tachycardia, and serous to mucopurulent nasal and ocular discharges occur and dyspnea and cough (sometimes up to 30%) are present in some pigs. Diarrhea, sometimes dysentery, and vomiting occur in some outbreaks and pregnant sows usually abort. Purple discoloration of the skin may be present on the limbs, snout, abdomen and ears. Abortion may occur in all stages of gestation about 5–8 days after the infection commences or after 1–2 days of fever. Death usually occurs within a day or two after the appearance of obvious signs of illness, and is often preceded by convulsions.
Historically, African swine fever was an acute to peracute disease with a case–fatality rate of almost 100%. However later, subacute and chronic diseases were observed. More recently, an even less virulent form of the disease has evolved. High fever and varying degrees of depression and lethargy are observed during the acute phase but some pigs continue to eat, case–fatality rate is usually less than 5%, the fever subsides in 2–3 weeks and the pigs return to full feed and grow at a normal rate. Recovered pigs have no lesions suggestive of the disease but may be viremic for several weeks. These persistently infected pigs would pass routine antemortem inspection at slaughter and potentially infectious offal and carcass trimming could be fed unknowingly to other pigs. Chronic cases are intermittently febrile, become emaciated and develop soft edematous swellings over limb joints and under the mandible.
Diagnosis depends on clinical signs (which are not distinguishable in the field from acute PDNS or CSF), post-mortem examination (it is said that button ulcers and turkey egg kidney are more rare in ASF but this cannot and must not be relied upon) but most importantly on diagnostic tests to rule out CSF and confirm ASF.
As in hog cholera, there is a fall in the total leukocyte count to about 40–50% of normal by the fourth day of fever. There is a pronounced lymphopenia and an increase in immature neutrophils. In chronic cases there is hypergammaglobulinemia. Clotting times are increased from about 5 days post-infection. Thrombocytopenia is detectable from day 6.
Antigen can be detected by the fluorescent antibody technique in tonsil and mandibular lymph node within 24–48 hours of infection and elsewhere once generalization has occurred. The indirect fluorescence antibody and direct fluorescence tests are commonly carried out on pooled visceral fluid samples.15
Antibody to the virus may be detected within 7 days of infection. The ELISA is highly sensitive and specific and can be automated for screening large numbers of sera. It has been developed for a variety of ASF proteins such as p73 or p30. More than 90% of infected pigs can be detected by the demonstration of specific antibodies against the virus. An immunoblotting assay is a highly specific and sensitive test which is easy to interpret, and provides an alternative to immunofluorescence and can be carried out in less than 90 min under field conditions.65 Complement testing is also a possibility. The inadequate storage or transport of sera may lead to samples being kept at high temperatures for long periods and up to 20% of these may be false-negatives by ELISA. All blood samples should be held at 4°C before testing and if incorrectly stored or handled should be tested by immunoblotting.66 A monoclonal antibody immunoperoxidase test is also useful for screening purposes.
Gross changes at necropsy resemble closely those found in hog cholera except that in the acute ASF, the lesions are more severe. In many organs there is a hyperemia or edema, with fibrinous microthrombi.67 The most common gross findings are swollen and hemorrhagic gastrohepatic and renal lymph nodes, often so badly affected that they may resemble the spleen, subcapsular petechiation of the kidneys, ecchymoses of the cardiac surfaces and various serosae, and pulmonary edema with hydrothorax.50 There may be hemopericardium. The renal hemorrhages are considered almost pathognomonic and are a consistent lesion following inoculation of pigs with the virulent or moderately virulent virus.57 Splenomegaly is usual but in contrast to hog cholera, splenic infarcts are rarely seen. The gallbladder is edematous and hemorrhagic but this is not as sometimes thought a pathognonomic lesion. In chronic cases the lesions are essentially the same but also include pericarditis, interstitial pneumonia and lymphadenitis. There is severe submucosal congestion in the colon, although button ulcers in the large intestine are less common than in hog cholera. Histologically the lesions are more diagnostic. The virus causes destruction of the mononuclear phagocyte system and then infects megakaryocytes, tonsillar crypt cells, renal cells, hepatocytes and endothelial cells. Postcapillary venules undergo hyalinization and endothelial swelling. Destruction of monocytes/macrophages is visible in the lymph nodes, the spleen and the bone marrow. In the liver, there is extensive destruction of hepatocytes. Marked karyorrhexis of lymphocytes is visible in both normal lymphoid tissues and in the infiltrating population of cells within parenchymatous organs. An encephalitis may be present with lymphoid infiltration of the leptomeninges, but is generally less severe than that of hog cholera. In recovered animals the presence of virus and antibody simultaneously (persistent infection) can cause the formation of immune glomerulonephritis.
As for hog cholera, the diagnostic testing to confirm ASF tends to be restricted to specialized laboratories.
• Histology – formalin-fixed spleen, lung, lymph nodes, kidney, liver, colon, cecum, brain (LM). The virus can be detected by IHC or ISH24
• Virology – spleen, kidney, submandibular and abdominal lymph nodes, tonsil (ISO (the virus grows well in bone marrow), FAT, PCR). A highly sensitive PCR has been developed.68 A Taqman PCR has been developed for ASF.69 A PCR has also been developed that can be used on a blood sample on filter paper70 and also a plaque assay.71 The PCR developed using the p72 protein will enable detection of ASF within 5 hours of clinical sample submission and full characterization of the virus within 48 hours.5
The disease is easily confused with hog cholera and very careful examination is required to differentiate the two. Clinically, the illness is much shorter (2 d as against 7 d) than in hog cholera. Gross necropsy changes are similar to but more severe than those of hog cholera. The marked karyorrhexis of lymphocytes characteristic of ASF is not observed in hog cholera. Differential diagnosis must rely on laboratory testing. In the past, differentiation has been achieved by the challenge of hog cholera-susceptible and immune pigs with suspect material. More recently, reliance has been placed on the demonstration of hemadsorbing activity with virus from suspected outbreaks grown on pig leukocyte tissue cultures. But hemadsorbing activity may be weak, delayed or even absent and there is sometimes difficulty in isolating virus from subacute or chronic cases in enzootic areas. Demonstration of antigen by fluorescent antibody staining will allow diagnosis of acute cases. For chronic cases, serological testing has been recommended and with the use of more than one test a high degree of accuracy can be achieved. Several sensitive laboratory tests for detection of the virus in tissues and serum antibody are now available. ELISA tests are highly sensitive. Radioimmunoassay tests are also sensitive and isolates of the virus may be titrated in swine monocyte cultures using a microtechnique. In the lymphocyte response test to virus infection, there is a cytolytic effect on the lymphocytes; the effect is greater on the B-lymphocytes than on the T-lymphocytes. Pigs with demonstrable antibody should be considerred as chronic carriers of the virus as it is doubtful that true recovery ever occurs.
Slaughter affected pigs and their ticks as quickly as possible.
The control and eradication of ASF is difficult because of the:
• Lack of an effective vaccine
• Transmission of the virus in fresh meat and cured pork products
• Recognition of persistent infection in some pigs, particularly wild feral pigs, possibly warthogs and bush pigs
• Clinical similarity of hog cholera and African swine fever
• Recognition that in some parts of the world soft ticks of the genus Ornithodoros (erraticus, moubata, porcinus porcinus) are involved in the biological transmission of the disease and can remain carriers for long periods (possibly 5 years).
Prevention of introduction of the disease to free countries is based on the prohibition of importation of live pigs or pig products from countries where African swine fever occurs. Strict application of the prohibition has prevented the spread of the disease from enzootic areas within South Africa. If a breakdown does occur, control must consist of prevention of spread by quarantine, slaughter of infected and in-contact animals and suitable hygienic precautions. The need for close contact between pigs for the disease to spread and the ease with which this can be prevented by the erection of pig-proof fences facilitates control. Conversely, the disease is virtually uncontrollable when pigs from a number of farms have access to communal grazing.14 The virus is highly resistant to external influences including chemical agents and the most practical disinfectant to use against the virus is a strong solution of caustic soda. Contaminated sties can remain infective for periods exceeding 3 months. These factors and the persistence of the virus in recovered pigs probably contributed to the difficulties encountered in the eradication program in Portugal, where the disease was stamped out but reappeared in 1960. However, the most important factor appears to have been the indiscriminate use of attenuated vaccines, which fostered the development of carrier pigs. In this outbreak so very little was seen in the form of clinical signs.
In Spain in 1985, a comprehensive nationally coordinated program for the eradication of the disease was begun and substantial progress had been made.72,73 Prior to 1985, the only method of control of the disease in Spain was depopulation of herds with clinical disease. The current eradication program consists of the following:
• Depopulation of herds with clinical disease
• Serological surveillance of all sows and boars in every herd
• Improvement of sanitary conditions of housing
• Improved hygiene (safe disposal of manure, vehicle disinfection, insect and rodent extermination)
• Veterinary control of all swine livestock transfers (with individual identification of every animal moved for finishing or breeding purposes)
• Health certification of every animal used for herd replacement
• Destruction of every seropositive animal
• Formation of mobile veterinary field teams exclusively dedicated to support the program.
Following introduction of this program, it has been possible to divide Spain into a disease-free region (the criteria is a minimum of 2 years without the disease) and an infected region. Eradication of the disease in Spain occurred by 2001.73 In 1991, the Spanish government claimed that 96% of the Spanish territory was free of ASF.73 The calculated benefit–cost ratio is estimated to vary from 1.23 to 1.47, depending on the intensity of the program. A reduction in the funding for control would result in a benefit–cost ratio of 0.97, making the program unprofitable.
Several different vaccines have been used, including an ineffective inactivated virus vaccine and modified live virus vaccines. The modified live virus vaccines provide some protection but the results following their use have been neither satisfactory nor safe and they have the two disadvantages of confounding laboratory tests and producing ‘carrier’ pigs.
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Geering WA, et al. Manual on procedures for disease eradication by stamping out. FAO Animal Health Manual 2001; No 12, 130 pp.
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Arias M, Sanchez-Vizcaino JM. African swine fever. In: Morilla A, Yoon K-J, Zimmermann J, editors. Trends in emerging viral diseases of swine. Ames: Iowa State Press; 2002:119-124.
Arias M, Sanchez-Vizcaino JM. African swine fever eradication; the Spanish model. In: Morilla A, Yoon K-J, Zimmermann J, editors. Trends in emerging viral diseases of swine. Ames: Iowa State Press; 2002:133-139.
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Hernaez B, Escribano JM, Alonso C. Switching on and off the cell death cascade; African swine fever virus apoptosis regulation. Prog Mol Subcell Biol. 2004;36:57.
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Worldwide distribution. Affects all species of Equidae. Transmission of disease by contaminated blood of clinically affected or inapparently infected horses during interrupted feeding of blood-feeding insects. Horses are infected for life
Fever, depression, edema, petechial hemorrhages, abortion, chronic weight loss, splenomegaly
The virus causing equine infectious anemia (EIAV) is a retrovirus, a member of the subfamily Lentivirinae of the family Retroviridae. The virus is an RNA virus that uses a reverse transcriptase enzyme to generate proviral DNA which is spliced into the host’s genome. The virus infects only Equidae and there is not evidence that it infects or causes disease in humans. EIAV shares antigenic cross-reactivity with human and feline immunodeficiency viruses but not with the viruses causing caprine arthritis–encephalitis or maedi–visna of sheep. The virus has one major group-specific antigen, p26, that is conserved and is the basis of the agar gel immunodiffusion (AGID) and competitive ELISA diagnostic tests. There is considerable antigenic drift in the surface glycoproteins (gp45, gp90) and the emergence of novel antigenic strains within an individual horse is associated with the relapsing febrile reactions characteristic of the disease. Examination of variations in the viral regulatory protein, Rev, and the transmembrane protein, gp90, demonstrates the existence of viral quasispecies such that genetically distinct viral subpopulations of differing phenotype exist within a chronically infected, often asymptomatic, animal.1,2 Mutations in gp45 and gp90 are random and related to the lack of a proof reading capacity of the viral reverse transcriptase enzyme.3
EIA has been diagnosed on all continents except Antarctica. In Europe it is most prevalent in the northern and central regions. It has appeared in most states of the United States and the provinces of Canada but the principal enzootic areas are the Gulf Coast region and the northern wooded sections of Canada.4
Extensive serological surveys using the AGID (Coggin’s) test have shown rates of infection of 1.5–2.5% in the United States, 6% in Canada, a low level in France, 1.6% in West Germany and 15–25% in Argentina. Within a geographic area, the prevalence of infection (positive AGID) varies depending on the density of the population, the proportion of carrier animals and the density of the population of insect vectors. Under ideal conditions the incidence of infection can approach 100% over a period of weeks, but this rapid spread is unusual.5
The morbidity varies considerably and depends on the strain of the virus and the inoculum delivered by the biting insect. Some horses become acutely ill and die after infection, while in others the infection is clinically inapparent. Outbreaks of disease associated with EIAV in horses of developed countries are rare.
Horses and ponies are susceptible to infection by EIAV and characteristically develop signs of the disease within days to weeks of infection. Mules also become infected and develop clinical signs similar to that of horses and ponies when infected with strains of the virus pathogenic to horses, but donkeys do not subsequently develop signs of the disease despite persistent infection with the horse-derived virus.6,7 The resistance of donkeys to horse-derived strains of EIAV is not definitive evidence that donkeys do not develop equine infectious anemia and there is suspicion that strains of the virus pathogenic to donkeys exist.7
The source of all new infections of EIAV is an infected horse, donkey or mule. Horses are persistently infected and clinically normal infected horses are a source of the virus.8 The virus can also be spread from clinically affected animals which, because of the high concentration of virus in their blood, are a potent source of infection and important in the rapid spread of infection. Transmission of EIAV occurs almost exclusively through the transfer of contaminated blood or blood products. In field conditions this occurs through the mechanical transmission of contaminated blood from an infected horse to an uninfected horse by biting insects.
The insect vectors responsible for the transmission of EIAV between horses are all large biting flies including Stomoxys calcitrans (stable fly), Chrysops sp. (deer fly), and Tabanus sp. (horse flies). Mosquitoes are not recognized as an important vector. Transmission is mechanical because the virus does not replicate in insects and is related to the large (10 nL) quantity of blood that the biting insects are capable of holding in their mouth.9 Infection occurs only if the feeding of the insect is interrupted. If this occurs the insect may attempt to feed again on the initial host or may seek another host that is close by. If the initial host is infected, the insect can carry blood from this animal to the second host and spread the infection. Tabanid flies can travel over a distance of 6 km, but when feeding is interrupted, the flies usually attempt to complete the meal on the initial host or a nearby animal and rarely travel more than 200 m to complete the meal.10
Insect factors that influence the likelihood of spread include11:
• Climate and season (tabanids prefer hot and humid conditions for feeding and breeding and their activity is much reduced or absent in winter months)
• Attractiveness of the host (foals are less likely to be bitten)
• Proximity of hosts to woodlands (tabanids preferred treed or sheltered habitat)
• Host housing (tabanids do not enter closed shelters)
• Distance between horses (as noted earlier, tabanids prefer to complete an interrupted meal on the initial host or a nearby host).
Intrauterine infection can occur and result in abortion or the birth of infected foals that often die within 2 months.11 Mares can be infected by insemination with semen containing the virus. Infection can be readily achieved by the use of contaminated surgical instruments or needles or by the injection of minute quantities of virus, and the use of a common needle when injecting groups of horses can cause an outbreak of the disease. In enzootic areas outbreaks have been caused by the use of untreated biological preparations of equine origin.
The virus is also capable of invasion through intact oral and nasal mucosae, wounds and even unbroken skin, but these portals are probably of minor importance in field outbreaks. Transmission of infection from horse to horse seems possible via swabbing instruments used to collect saliva for doping tests.
After infection, EIAV multiplies in tissues that have abundant macrophages with the spleen being the principal site of viral infection and propagation and accounting for over 90% of cellular viral burden.12 Viral replication occurs only in mature tissue macrophages and circulating monocytes account for only 1% of the cellular viral burden.12 The concentration of cell free virus in blood, which can be as high as 106 TCID 50% per mL, parallels the clinical course. Fever and other clinical signs develop within 2–7 d of infection as the concentration of virus in the blood increases, and resolves as the viremia abates. There is a persistent but low level viremia that persists for the life of the horse. The level of viremia in horses without clinical signs of the disease is very low and undetectable using conventional virus culture techniques but evident using PCR.13 The virus is detectable in low concentrations in most tissues of asymptomatic horses.12 During periods of relapse of the clinical disease the degree of viremia increases. On these occasions, the virus isolated from the blood has antigenic characteristics different than that which originally infected the horse. Antigenic drift of the gp45 and gp90 antigens, which occurs constantly even in asymptomatic horses with low levels of viremia, allows mutations of the virus that then avoid immune surveillance, multiply and cause clinical disease.2 The frequency of relapses of the clinical disease declines markedly after the first year of infection and horses that survive become asymptomatic carriers.
The immune response to EIAV is responsible for controlling replication of the virus and also plays an important role in the pathogenesis of the disease. The major clinical signs and lesions of equine infection anemia are attributable to the host response to the virus and not direct viral damage to tissue.3 Replication of EIAV stimulates a strong immune response that is detectable in horses and ponies within 7–10 d of infection. The initial infection is likely controlled by cytotoxic T-lymphocytes before the appearance of neutralizing antibodies.13 Antibodies to the p26 core protein are detectable by AGID test in almost all horses 45 d after infection and by 60 d after infection antibodies to gp45 and gp90 are present. The neutralizing antibodies are specific to the phenotype of virus causing the viremia – this phenotype can change over time as discussed above. Hypergammaglobulinemia develops. The immune response includes the production of virus neutralizing antibodies, complement-fixing antibodies, and cytotoxic T-lymphocytes.13 The immune responses are responsible for the termination of viremia although this effect is not mediated by antibody-dependent cellular cytotoxicity against EIAV-infected macrophages,14 but rather by development of neutralizing antibody and cytotoxic T-lymphocytes.13 The importance of neutralizing antibodies in control of the disease within an animal is indicated by the observation that viremia is never associated with a virus with a neutralizing phenotype already recognized by the horse.13
Most virus in viremic horses is a complex of virus and antibody. The virus-antibody complex is readily phagocytosed by cells of the reticuloendothelial system, including tissue macrophages, and is involved in the development of the fever, depression, thrombocytopenia, anemia and glomerulonephritis characteristic of the disease.15
Neurologic disease in horses with EIAV infection is attributable to viral infection of neural tissue but not necessarily neurons.15
The anemia characteristic of horses experiencing several febrile episodes of EIA is attributable to shortened life of red blood cells and decreased red cell production. Infection with EIAV shortens the lifespan of circulating red blood cells to about 38 d, compared with the normal value of 130 d.16 The reduction in red blood cell lifespan is likely due to the presence of virus–antibody complexes on the surface of red blood cells with subsequent clearance of such cells by the reticuloendothelial system as evidenced by the presence of sideroleukocytes in peripheral blood of infected horses. EIAV also has a suppressive effect on erythroid series cells in bone marrow.17 Anemia occurs in Arabian foals with severe combined immunodeficiency infected with EIAV, indicating that the anemia is not wholly due to the adaptive immune response of the host.18 Anemia of chronic disease, which is due in part to limited availability of iron stores, likely contributes to the lack of bone marrow response.
Thrombocytopenia is a consistent feature of the acute, febrile episodes of EIA and has been attributed to the deposition of virus–antibody complexes on platelets with subsequent removal of affected platelets by tissue macrophages.19 However, others have identified a primary production deficit due to an indirect, non-cytocidal suppressive effect of EIAV on megakaryocytes.20 EIAV does not infect megakaryocytes and the suppressive effect of infection is due at least in part to tumor necrosis factors alpha and beta.20,21 Another explanation for the thrombocytopenia is increased removal of platelets because of increased in vivo activation and formation of platelet aggregates, a form of non-immune mediated platelet destruction.22 This was associated with increased thrombopoiesis and an increase in the proportion of young platelets in blood.22 The precise mechanisms underlying the thrombocytopenia associated with acute EIAV infection of horses is unclear.
Platelets of EIAV infected horses with clinical signs of disease have diminished function in vitro.22 Platelets from infected horses had greater amounts of fibrinogen bound to their surface, ultrastructural abnormalities consistent with activation, and diminished in vitro aggregation responses.22
The cell reservoir of virus in persistently infected horses is unknown, as are the mechanisms underlying latency. However, the ability of retroviruses to splice a DNA copy of their genome into the genome of the host is probably important in the persistence of viral infection. Viral genome is detectable in clinically normal but persistently infected horses.6 Presence of viral DNA in host tissue is indicative of infection whereas the presence of viral RNA in blood is suggestive of viral replication.6 This viral strategy allows the virus to escape immune surveillance of the host. Factors triggering a relapse of virus production from the latent genome are unknown, but relapse is associated with antigenic drift.
A likely scheme of pathogenetic events includes:
• Primary entry and infection of tissue macrophages, especially in the spleen
• Destruction of macrophages and release of virus and components
• Production of antibodies to antigenic components
• Formation of antigen–antibody complexes, which induce fever, glomerulitis, anemia, thrombocytopenia and complement depletion
• Hemolysis or phagocytosis caused by specific complexes activating the reticuloendothelial system
• Temporary iron-deficient erythropoiesis caused by delayed release of iron from macrophages
• Subsidence of pathological processes as virus-neutralizing antibody restrains viral multiplication in macrophages. The virus is incorporated into the host genome and becomes latent
• Appearance of a new antigenic variant of the virus and commencement of a new cycle of viral replication in macrophages and a new clinical episode. The antigenic variation is due to changes in the surface glycoprotein of the EIA virus
• Less frequent recurrence of these episodes and the horse becomes permanently asymptomatic. The animal can be said to have achieved an appropriate level of immune response sufficient to protect it against antigenic epitopes that are common to all EIA virus strains.
An incubation period of 2–4 weeks is usual in natural outbreaks of equine infectious anemia. Outbreaks usually follow a pattern of slow spread to susceptible horses after the introduction of an infected animal. On first exposure to infection, horses manifest signs of varying degree, classified as acute or subacute. Occasionally the initial attack is mild to inapparent and may be followed by rapid clinical recovery. As a rule there is initial anorexia, depression, profound weakness and loss of condition. Ataxia, behavioral changes, hyperesthesia and blindness occur and in some horses is recorded as the only clinical abnormality.18,23. There is intermittent fever (up to 41°C; 105°F) which may rise and fall rapidly, sometimes varying as much as 1°C within 1 h. Jaundice, edema of the ventral abdomen, the prepuce and legs, and petechial hemorrhages in the mucosae, especially under the tongue and in the conjunctivae, may be observed. Pallor of the mucosae does not occur in this early stage and they tend to be congested and edematous. There is a characteristic increase in rate and intensity of the heart sounds, which are greatly exacerbated by moderate exercise. Respiratory signs are not marked and there is no dyspnea until the terminal stages, but there may be a thin serosanguineous nasal discharge. There is considerable enlargement of the spleen which may be detectable per rectum. Pregnant mares may abort. Many animals recover from this acute stage after a course of 3 d to 3 weeks. Others become progressively weak, recumbent and die after a course of 10–14 d of illness.
Animals recovering from the acute disease may appear normal for 2–3 weeks and then relapse with similar but usually less severe signs. Death may occur during such a relapse. Relapses continue to occur at intervals of as little as 2 weeks but usually cease after about a year. If they recur, they are usually associated with periods of stress and characterized by fever, increasing emaciation, weakness, ventral edema, and the development of pallor of the mucosae, a late sign of this disease. In this chronic stage, the appetite is usually good, although allotriophagia may be observed. Some affected animals appear to make a complete recovery but they remain infected and may suffer relapses in later years. Prolonged therapy with corticosteroids can cause such a relapse. Even in the absence of clinical illness infected animals often perform less efficiently than the uninfected. Most deaths occur within a year of infection. Survivors persist as asymptomatic carriers.
Hematological examination of horses with the acute disease reveals a moderate to marked thrombocytopenia and an anemia that may be severe. Thrombocytopenia occurs during relapses of the disease, is most severe during the febrile episodes, and may be sufficiently low that it allows petechial hemorrhages to develop. The anemia may become more severe with relapse (14–20%, 0.14–0.20 L/L) and is normocytic and normochromic. The presence of sideroleukocytes (leukocytes containing hemosiderin) are considered highly suggestive of EIA.24 There are no characteristic changes in the white blood cell count.
Hypergammaglobulinemia may be present. Serum biochemical examination may reveal an increase in bilirubin concentration and a decrease in serum iron concentration.
Diagnostic confirmation is achieved through detection of antibodies to the p26 core antigen of EIAV. Two tests are in general use: the AGID test (Coggin’s test) and a number of ELISAs including a competitive ELISA (CELISA) test.25,26 Results of AGID testing are available in 24 hours while those of ELISA testing can be available in as little as one hour. Two commercially available ELISA tests detect antibody to the p26 antigen whereas the other detects antibody to the gp45 transmembrane protein. The ELISA tests inherently have greater sensitivity (detect lower concentrations of antibody) than does the AGID but often the characteristics of the commercial ELISA assays are modified to decrease the sensitivity (increase the lowest concentration of antibody detected by the kit) so that results obtained with these kits are concordant with those obtained by AGID.25 The ELISA for detection of antibodies to gp45 has a slightly lower sensitivity than do those that detect antibody to p26.27 For practical purposes the tests may be considered to have equal sensitivity and specificity, and are very accurate in the diagnosis of EIA.26 However, the CELISA test can detect lower concentrations of antibody than the AGID can, therefore suspected false-negative or equivocal tests based on the AGID can be repeated using the CELISA.26 Conversely, the CELISA has a slightly higher false-positive rate than the does the AGID, and positive reactions on the CELISA should be verified by AGID. A positive AGID test is accepted as synonymous with being infected and infective.
An advantage of an ELISA that detects antibodies to gp45 antigen is that, when combined with testing for antibodies to p26, the ability to detect infected horses with equivocal test results on a single test is increased.25 This is similar to the technique of using a Western blot test to demonstrate the presence of antibodies to more than one antigen, especially those against the gp45 and gp90 antigens, when equivocal AGID or ELISA results are obtained.
False-negative reactions for either test may occur because the horse lacks antibodies to the p26 antigen. The AGID and CELISA tests might not detect a recently infected horse that has yet to develop antibodies. Some horses do not develop anti-p26 antibodies for 45 d after infection. False-positive reactions may occur in foals born to infected mares. Colostral transfer of anti-p26 antibodies to the foal will be detectable up to 6 months after birth.
Positive reactions to ELISA testing (to the p26 antigen) in samples that are negative by AGID testing can be the result of interspecies determinants.26 It is suspected that horses exposed to related lentivirus produce antibodies to structural proteins that cross react with the EIAV p26 antigen in ELISA, but not AGID, testing.
An algorithm for testing of equine samples for EIAV is provided in Table 21.1.
Table 21.1 Algorithm for testing horses for infection by equine infectious anemia virus when the prevalence rate is less than 1 in 100024
Tests to detect proviral DNA or viral RNA in blood and tissue have been developed and are useful in detecting the presence of virus when viral concentrations in blood and/or tissue are low.28,29 The identification of proviral DNA in blood of infected horses is as specific and apparently more sensitive than the AGID in detecting infected horses.29
Experimental transmission of the disease to susceptible horses by the SC injection of 20 mL whole blood or Seitz-filtered plasma is also used as a diagnostic test and is a valuable, although expensive and archaic supplement to other tests. The donor blood should be collected during a febrile episode when the viremia is most pronounced, and the recipient animals are checked for increases in body temperature twice daily.
In the acute stages, there may be subcutaneous edema, jaundice and petechial or ecchymotic subserosal hemorrhages. There is considerable enlargement of the liver and spleen, and local lymph nodes. The bone marrow is reddened due to increased amounts of hematopoietic tissue and may contain focal infarcts. In the chronic stages, emaciation and pallor of tissues are often the only gross findings. Histological examination is helpful in the diagnosis, even in asymptomatic chronic carriers. Characteristic lesions include hemosiderosis, perivascular infiltrates of round cells in many organs, and an extensive proliferation of the mononuclear phagocytic cells throughout the body. A glomerulitis, probably caused by the deposition of virus–antibody complexes on the glomerular epithelium, may be present. Lesions in the brain are a lymphohistiocytic periventricular leukoencephalitis.15 Culture of this virus is time-consuming and the diagnosis is usually confirmed on the basis of a positive serologic test and typical microscopic lesions.
No specific treatment is available. Supportive treatment including blood transfusions and hematinic drugs may facilitate clinical recovery but it is important to remember that recovered horses are persistently infected and infectious for life.
Control of EIA is based on identification and eradication or life-long quarantine of infected animals, quarantine and testing of new stock, compulsory testing of imported horses, and efforts to prevent spread of the virus by controlling insect access to horses and use of strict hygiene when vaccinating or collecting blood samples from horses.
The control of equine infectious anemia is still universally based on the eradication of the disease by identifying the infected, clinically normal animals with a serological test and then destroying them. Identification is by means of the AGID or CELISA tests. The ability of a program of test and eradication to eliminate the disease is evidenced by the eradication of EIA from Hong Kong. An effective control program is described for Kentucky in the United States that permits the maintenance of infected horses with indelible identification and prescribed restrictions on housing.30
Control programs based on this test-and-slaughter policy are under fire because of the view of horse owners that many asymptomatic horses, with very low infectivity, are being destroyed unnecessarily. A decision on the matter depends on whether the objective is eradication or containment, and if the latter, at what level. Until now the policy has been eradication and it is obvious that another attitude is possible. Some flexibility in official attitude is desirable because of the fallibility of the recommended control procedures and the devastating losses that can occur when the optimum environment for the spread of the disease is encountered.
Foals from infected mares can be raised free of infection.31 Such foals have detectable antibodies to EIAV for up to 330 days on immunoblot and 260 days on ELISA testing because of transfer of maternal immunoglobulins in colostrum during the neonatal period. However, foals that are ultimately free of infection do not have detectable viral RNA in blood and have declining concentration of antibodies to EIAV.31 Foals should be isolated from infected horses as soon as feasible after diagnosis of EIAV infection in the dam.
Restriction of introduction of infected horses into clean herds or areas is important to prevent introduction of the disease. Horses should be tested before introduction to the herd, and perhaps again in 1 to 2 months. If suspect horses are to be introduced, they should be kept under close surveillance for at least 6 months before being admitted. Horses known or suspected to be infected should be separated from all other uninfected horses, donkeys and mules by a distance of at least 200 m. This recommendation is based on observations of the feeding behavior of tabanids, which are very unlikely to fly more than 100 m after an interrupted feeding. Suspect positive horses should be retested after at least 1 month and probably at regular intervals thereafter. Operators of open stud farms, and rest farms can also insist on proof of a negative test before admitting each horse. One deficiency of this policy is the long period of ‘incubation’ of up to 45 d between infection and seroconversion to a positive test.
In countries where the incidence is high, it is usual to control horse movement by a system of permits and certificates of freedom from the disease, and to insist on skin branding or lip tattooing of all horses. AGID or ELISA test-positive horses should be allowed to move only under specified conditions.26
Draining of marshy areas and the control of biting insects may aid in limiting spread of the disease. A degree of protection may be obtained by the use of insect repellents and by stabling in screened stables. Great care must be taken to avoid transmission of the disease on surgical instruments and hypodermic needles, which can only be sterilized by boiling for 15 min or by autoclaving at 6.6 kg pressure for a similar period. Chemical disinfection of instruments and tattoo equipment requires their immersion for 10 min in one of the less corrosive phenolic disinfectants. All materials to be disinfected need to be cleaned of organic matter first. For personal disinfection sodium hypochlorite, ethanol or iodine compounds are safe, and for materials where organic matter is not removable, agents such as chlorhexidine or phenolic compounds combined with a detergent are satisfactory.
There are considerable problems associated with development of vaccines against EIA because only neutralizing antibodies are capable of causing sterile immunity and preventing infection.13 Neutralizing antibodies are specific for the homologous virus, but the large variation in phenotypes of the wild virus means that it will be difficult to stimulate neutralizing antibodies protective against all of the possible infecting phenotypes. Vaccines are available in parts of the world but are not in general use. Killed, whole virus vaccines are safe but subunit vaccines may actually enhance the occurrence of the disease.32 An experimental live attenuated EIAV DNA proviral vaccine affords complete protection in experimentally infected horses but is not commercially available at the time of writing.33
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